Hemorrhagic enteritis virus DNA sequences, proteins encoded thereby and various uses thereof

Abstract

The present invention relates to a DNA sequence comprising a nucleotide sequence encoding Hemorrhagic Enteritis virus. It is well known to the man of the art that determining the complete sequence of a virus enables the isolation and identification of the different genes contained therein, and their utilisation for different purposes such as for vaccination purposes, as potential vectors for gene delivery to be used in recombinant vaccination or for gene therapy. In addition, the sequence may be employed for diagnostic purposes wherein the disclosed sequence of any part thereof be used for the development of specific primers for Polymerase Chain Reaction processes (PCR) or as probes. The invention thus also concerns with HEV proteins encoded by the sequence of the invention or functional fragments thereof and to some of the uses of said sequences and proteins.

Description

FIELD OF THE INVENTION

The present invention relates to a DNA sequence which comprises a nucleotide sequence encoding the Hemorrhagic Enteritis Virus, to proteins encoded thereby, to vectors and DNA constructs comprising the said DNA sequence or essential fragments thereof, and to various uses of the DNA sequence and the proteins encoded thereby.

BACKGROUND OF THE INVENTION

One of the principal diseases which suppress the immune system in turkeys is caused by infection with Hemorrhagic Enteritis Virus (HEV). HEV belongs to the Adenoviridae family. This family consists of serotypes that infect mammals (Mastadenoviridae) and avian (Aviadenoviridae) [Shenk T. Virology, pp. 2111-2148 (1996), B. N. Fields, D. M. Knipe and P. M. Howley (Eds) Lippincott-Raven New York]. HEV, together with marble spleen disease of pheasant and splenomegaly virus of chickens, are classified as type II avian adenovirus (Ad) [Domermuth C. H. and Gross W. B., Diseases of Poultry 8th ed. Pp.511-516, H. J. Barnes, B. W. Calnck, W. B. Reid and Yoder H. W. (Eds) Iowa State University Press (1984)] which is serologically distinct from type I and type III isolated from chickens infected by fowl Ad (FAV) 1-12 and egg drop syndrome (EDS) virus, respectively.

The HEV, as other type II Ad's, is a non-enveloped DNA virus, with a diameter of about 70-90 nm and an icosahedral symmetry. The genome is linear, double-stranded DNA and with a size estimated to be approximately 25.5 kb [Jucker et al J. Gen. Virol. 77:469-479 (1996)]. Partial sequence (about 4 kb) of the HEV genome has been recently published [Jucker et al. (1996) ibid.], while full sequences of several human Ad's (types 2, 5, 12 and 40), avian Ad's (CELO and egg drop syndrome (EDS)) and ovine Ad were published and may be found in EMBL and GenBank data bases. The e organization of all human Ad's is very similar [Shenk T. (1996) ibid.]. However, in some non-human Adenovirus (Ad) sequences (CELO, Ova and EDS) no similarity was found for various regions of human Ad [Chiocca, S., et. al. J. Virol. 70:2939-3949 (1996); Vrati, S., et. al Virology 220:186-199 (1996); Hess, M. et. al., Virology 238:145-156 (1997), respectively].

HEV replicates in the host cell nucleus and consists of 11 proteins, encoded by its DNA segment. The molecular weights of the HEV proteins range from 14 kD to 97 kD [Nazerian K. L., et al. Avian Dis. 35:572-578 (1991)]. The 97 kD polypeptide is the structural hexon protein, a monomer of the major outer capsid. Other structural proteins are the penton base protein, having a predicted size of about 50 kD and the fiber protein which anchors the penton base protein. This fiber protein consists of a tail and a globular head, which plays an important role in the first attachment of the virus to the cell receptor.

The virus infects turkeys and causes a disease which is characterized by depression, splenomegaly, intestinal hemorrhages and immuno-suppression [Domermuth C. H., & Gross W. B. (1991) Diseases of Poultry, 9th Edition, M. S. Hofstad et al. Eds. Iowa State University Press, Ames, Iowa]. The virus replicates in B cells and macrophages [Suresh M. & Sharma J. M. J. Virol. 70:30-36 (1996)] and is concentrated in large amounts in the spleen. Since B cells play an important role in the primary immune response, afflicted birds suffer mostly from weight loss.

Infection of birds by the HEV is especially prevalent during the ages of 7 to 9 weeks [Domermuth C. H. & Gross W. B., Diseases of Poultry, Iowa State University Press, 8th Edition pp. 511-516 (1984)]. Younger birds are protected by maternal antibodies [Van den Hurk, J. V. Avian Dis. 30:662-671 (1986); Harris J. R. & Domermuth C. H., Avian Dis. 21:120-122 (1977); Fadly, A. M. & Nazerian K. Avian Dis. 33:778-786 (1989)]. The rate of mortality of infected birds is high and, since the immune response is damaged, the surviving birds exhibit high vulnerability to other diseases. Moreover, infection with HEV reduces the effectiveness of response to various vaccines. As a result of lowered resistance, an outbreak of a HEV infection may further lead to outbreaks of other diseases. Naturally, such events result in heavy financial loss to the breeders.

Infectious diseases in animals, and in particular in farm animals, are one of the most important economic factors in agriculture, for example, in the poultry industry. The minimalization of losses from diseases, by means of effective vaccines, plays a major part in achieving profit in today's intensive agricultural industry. The health of domesticated animals depends on management, on a proper vaccination system and on the availability of effective vaccines.

Since the price of a single farm animal is relatively low, the cost of production and delivery of the vaccine becomes critical. Naturally, if cost of the production of the vaccine is too high, its use will not be economically worthwhile.

In the last decade recombinant adenoviral vectors have become a subject for research as vectors in gene therapy [Kozarsky K. F. & Wilson J. M., Current Options in Genetics and Development 3:499-503 (1993)]. The complete sequence of the viral DNA is essential for enabling successful manipulation of the virus, for use in gene delivery. To date, recombinant Ad's have been employed in a variety of gene therapy applications as carriers of foreign genes, as obtained with vaccinia and fowlpox [Yamanouchi K. K., et al. The Veterinary Record 13:152-156 (1993); Boursnell M. E. G. et al Virology 265:18634-18642 (1990), respectively] and in sub-unit vaccination [Israel Patent Application No.122626].

One aim of the present invention is to construct a recombinant HEV. There are a number of advantages in utilizing an adenoviridae type transfection and expression system such as the HEV derived vector of the present invention. These viruses are easy to grow giving high titers, and both the virions and the viral genome are very stable. Very high levels of expression are possible since most of the macromolecular biosynthesis in adenovirus-infected cells is virus directed at late time points post infection. Proteins made in Ad-derived expression systems would be expected to have all the post-translational modifications that might be important in determining their functional and antigenic properties, and may thus be useful for therapeutic, diagnostic or vaccination purposes.

In addition, the Ad genome is relatively easy to manipulate by recombinant DNA techniques, allowing incorporation of foreign genes as large a 7.5 kb. The ability or recombinant Ad's to terminally transduce differentiated cells in vivo has made these vectors important candidates for many gene therapy applications [S. I., Michael et al. Gene Therapy 2:660-668 (1995)]. Evidently, knowledge of the complete sequence of the viral DNA is needed in order to perform the required manipulations in the sequence.

Although permissive infections are ultimately lytic, infected cells remain intact until relatively late in infection, making the collection of concentrated virus and virus specified intracellular proteins fairly visible.

SUMMARY OF THE INVENTION

The present invention relates to a DNA sequence which comprises (a) a nucleotide sequence substantially as shown in SEQ ID NO:1; or (b) a nucleotide sequence which corresponds to the sequence substantially as shown in SEQ ID NO:1 within the scope of the degeneracy of the genetic code; or (c) a nucleotide sequence which hybridizes, under conditions that allow such hybridization to occur, with the sequences according to (a) or (b) or with a fragment thereof.

The DNA sequence of the invention encodes a Hemorrhagic Enteritis Virus (HEV) and biologically functional homologues and fragments thereof, or a non-virulent HEV which is capable of infecting a host cell upon exposure thereto.

The invention also concerns with a DNA sequence comprising (a) at least one nucleotide sequence selected from the nucleotide sequences substantially as shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28 and SEQ ID NO:30 and functional fragments thereof, said fragment being other than the nucleotide sequences substantially as shown in SEQ ID NO's:6 and 14 and other than the nucleotide sequence comprising nucleotide bases 187 to 1358 of the nucleotide sequence substantially as shown in SEQ ID NO:22;or (b) at least one nucleotide sequence selected from the nucleotide sequence which corresponds to the above sequences, within the scope of the degeneracy of the genetic code; or (c) a nucleotide sequence which hybridizes, under conditions that allow such hybridization to occur, with the sequence according to (a) or (b) or with a fragment thereof.

In a second aspect, the invention relates to a vector comprising the DNA sequence of the invention or deletions, insertions, mutations, replacements or modifications thereof.

In addition, the invention relates to a DNA construct comprising the DNA sequence of the invention or deletions, insertions, mutations, replacements or modifications thereof and at least one therapeutic exogenous nucleotide sequence operably linked thereto, the DNA construct being capable of transfecting a host cell, upon exposure thereto, with the exogenous nucleotide sequence, the exogenous nucleotide sequence being a sequence which is not made or contained in the cell or is made or contained in the cell in defective form.

In yet a further aspect, the invention relates to a DNA construct for the expression of a therapeutic protein or peptide product in a host cell, comprising a vector of the invention and at least one exogenous nucleotide sequence operably linked thereto which encodes the protein or peptide product.

In another aspect, the invention concerns host cells transformed with a nucleotide sequence of the invention and with host cells transfected with the DNA constructs of the invention.

In yet an additional aspect, the invention relates to a protein or peptide expressed by the host cells of the invention, wherein the protein or peptide product is a therapeutic protein or peptide product which is not made or contained in the cell, or is a therapeutic protein or peptide product which is made or contained in the cell in abnormally low amount, or is a therapeutic protein or peptide product which is made or contained in the cell in defective form or is a therapeutic protein or peptide product which is made or contained in the cell in physiologically abnormal amounts.

Alternatively, the invention relates to a protein or peptide product comprising a sequence substantially as shown in SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31 or functional equivalents and fragments thereof, said fragment being other than the amino acid sequences substantially as shown in SEQ ID NO's 6 and 14 and other than the amino acid sequence comprising amino acids 63 to amino acid 454 of the amino acid sequence substantially as shown in SEQ ID NO 23.

The invention also relates to a vaccine for immunizing a domesticated animal against at least one specific antigen comprising the DNA construct of the invention or the protein of the invention, optionally further comprising pharmaceutically acceptable carriers, diluents and additives.

Pharmaceutical compositions comprising as active ingredient a therapeutically effective amount of the DNA construct of the or of the cells of the invention or of the therapeutic protein or peptide product of the invention are also within the scope of the invention.

Finally, the different uses of the nucleotide sequence of the invention or the DNA constructs of the invention or the proteins of the invention in the preparation of a vaccine or a pharmaceutical composition also constitute part of the invention.

DESCRIPTION OF THE FIGURES

FIG. 1 A schematic representation of the genome organization of HEV and a comparison to EDS and Ad2 genomes

Genes of the HEV are referred to by the nomenclature of the corresponding sites in other Ad's. Locations of predicted sequences are indicated by full boxes. ORF's starting with the codon for methionine (Met) and which encode an amino acid sequence having more than 100 residues, and with no homology to any known Ad ORF, are designated ‘ORF1’ to ‘ORF8’ and indicated by empty boxes. EDS and Ad2 sequences were pooled from GenBank (Accession Nos. Y09598 and J01917, respectively). The upper boxes designate genes found on the upper strand. Abbreviations: POL: DNA polymerase; pTP: precursor terminal protein; CBP: DNA binding protein; EP: endoproteinase.

FIG. 2 Alignment of the last 11 amino acid at the C-terminal of pVI of HEV with other Ad's

The alignment was obtained by the Pileup program which is part of the GCG software package (see Example 4). Amino acids identical in all five viruses are boxed in black. Amino acids which are identical or similar among some of the viruses are indicated by gray boxes.

FIG. 3 Amino acid sequence alignment of endoproteinase of five Ad's

The alignment was obtained by the ClustalX software (see Example 4). The pairwise and multiple alignment parameters were 6 for gap opening penalty and 0.1 for gap extension penalty. The sequences are ordered according to their similarity. The conserved residues of the active site are shaded gray. Asterisks designate identical residues in all sequences and a colon designates conserved substitutions. A dot indicates a position in which more than 50% of the residues are identical.

FIG. 4 Invert terminal repeats (ITR) of HEV

The ITR of HEV was compared with the ITR's of Ova ( FIG. 4 a ) and EDS ( FIG. 4 b ). The alignment was obtained by Bestfit program which is part of the GCG software package (see Example 4). The gap opening parameter of the program was set to 15 and the gap extenuation parameter to 1.

FIGS. 5A-1 , 5 A- 2 and 5 A- 3 HEV fiber amino acid organization

FIG. 6 HEV genes.

The 3′ end of HEV genes with the following 20 nucleotides wherein the stop codons are underlined.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a DNA sequence which comprises (a) a nucleotide sequence substantially as shown in SEQ ID NO:1; or (b) a nucleotide sequence which corresponds to the sequence substantially as shown in SEQ ID NO:1 within the scope of the degeneracy of the genetic code; or (c) a nucleotide sequence which hybridizes, under conditions that allow for such hybridization to occur, with the sequences according to (a) or (b) or with a fragment thereof The DNA sequence of the invention encodes for Hemorrhagic Enteritis Virus (EV). The genome of HEV, which consists of this sequence is a linear, double-stranded DNA in the size of approximately 25.5 kb (SEQ ID NO:1). Evidently, the complete sequence of the virus enables the isolation and identification of the different genes contained therein, and their utilization for different purposes such as for vaccination purposes, as potential vectors for gene delivery to be used in recombinant vaccination or for gene therapy. In addition, the sequence may be employed for diagnostic purposes wherein the disclosed sequence of any part thereof be used for the development of specific primers for Polymerase Chain Reaction processes (PCR) or as probes.

Any sequence which, at the nucleotide level, has a homology of about 95% or at least 95% to the sequence of the invention or any biologically functional homologues and fragments of the sequence may also constitute part of the invention. Such sequences preferably encode a non-virulent HEV which is capable of infecting a host cell upon exposure thereto. Thus, the invention also concerned with a non-virulent HEV encoded by the DNA sequence of the invention.

In the same aspect the invention relates to a DNA sequence comprising (a) at least one nucleotide sequence selected from the nucleotide sequences substantially as shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28 and SEQ ID NO:30; or (b) at least one nucleotide sequence selected from the nucleotide sequence which corresponds to the sequences substantially as shown in SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:16, SEQ ID NO:18, SEQ ID NO:20, SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28 and SEQ ID NO:30, within the scope of the degeneracy of the genetic code; or (c) a nucleotide sequence which hybridizes, under conditions that allow such hybridization to occur, with the sequence according to (a) or (b) or with a fragment thereof. further, the invention relates to functional fragments of the above sequences, being other than the sequences shown in SEQ ID NO:6 and 14 or other than the nucleic acid sequence comprising nucleotides 187 to 1358 of the nucleic acid sequence substantially as shown in SEQ ID NO:22, corresponding to SEQ ID NO:3 described in PCT/IL98/00609, incorporated herein by reference, or to derivatives thereof. The said sequence described in PCT/IL98/00609 corresponds to the sequence form nucleic acid 187 to nucleic acid 1358 within SEQ ID NO:22 of the present invention.

All of the said sequences are derived from the DNA sequence substantially as shown in SEQ ID NO:1 and encode, respectively, the proteins designated hereinafter as 52K, IIIa, core protein I (CPI), core protein II (CPII), pVI, endoproteinase (EP), 100K, pVIII, the complete fiber, IVa2, DNA polymerase (POL), pTP and the DNA binding protein (DBP). The therapeutic merits of these proteins and of the nucleotide sequences encoding them are well recognized by the man of the art. For example, identification of nucleotide and amino acid sequences of the HEV structural proteins (hexon (SEQ ID NO:14), penton base (SEQ ID NO:6) and fiber proteins) may be utilized in the preparation of a sub-unit vaccine against the virus. In addition, since the fiber protein ( FIGS. 5A-1 , 5 A- 2 and 5 A- 3 ) is responsible for the first attachment of the virus to the cell receptor, modifications thereof may be utilized in altering its specificity to different host cells.

Fusion proteins encoded by recombinant vectors comprising the nucleotide sequences of the invention or other sequences, preferably, other therapeutic sequences, are also within the scope on the invention. Thus, a DNA sequence comprising at least one first nucleotide sequence selected from the nucleotide sequences substantially as shown in SEQ ID NO:6, SEQ ID NO:14, SEQ ID NO:22 (corresponding, receptively, to the structural proteins PB, hexon and fiber proteins) and at least one second nucleotide sequence operably linked thereto and selected from the nucleotide sequences shown in SEQ ID NO:4, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12 and SEQ ID NO:20 which function as structural stabilizers, may be constructed.

In any case, the DNA sequences of the invention are genomic DNA or cDNA.

In a second aspect, the invention relates to vectors capable of delivering into a host cell an exogenous nucleotide sequence of interest which is operably linked to the vector. The vector comprises the nucleotide sequence of the invention or deletions, insertions, mutations, replacements or modifications thereof or functional fragments thereof. Such delivery means any infection or transfection of the exogenous nucleotide sequence into a specific host cell, preferably resulting in recombinant integration of the foreign DNA into the DNA genome of the host cell.

In addition, the invention relates to DNA constructs comprising a vector of the invention, which, as defined, contains the DNA sequence of the invention or deletions, insertions, mutations, replacements or modifications thereof, and further comprising at least one therapeutic exogenous nucleotide sequence operably linked thereto. The exogenous nucleotide sequence preferably is inserted into the vector in a reading frame, to enable the expression thereof, in a host. The exogenous sequence may be present in only one copy. However, several copies of the exogenous sequence, or its combination with other therapeutic foreign sequences may be inserted. Such sequences may be separated by initiation and termination sequences or they may form a single reading frame, thus producing a single “fusion protein”.

The DNA construct of the invention is a viral construct capable of infecting a host cell, upon exposure thereto, with the therapeutic exogenous nucleotide sequence which, under normal conditions, is not made or contained in the host cell or is made or contained in said cell in a defective form. Delivery into cells of linear DNA, by infecting the cells with constructs of the invention comprising such linear DNA, may be advantageous for recombination, i.e. integration into the cellular genome for stable expression.

The viral constructs of the invention which are in fact recombinant viruses, will comprise the essential features for infecting the desired host cell, and nevertheless be non-virulent.

Obviously, the viral constructs can also be modified with specific receptors or ligands to alter target specificity through receptor mediated events. Alternatively, a modified, selective targeting of the constructs is possible by producing a DNA construct according to the invention containing a modified fiber protein. It is known that alterations at the nucleotide level, i.e. mutations, insertions, or deletions or modifications at the protein level, may alter the specificity of the protein. For example, it has been shown [Stevenson S. C. et al. Virology 71(6):4782-4790 (1997)] that exchanging the head domain for other serotypes which recognize a different receptor, changes the specificity of the expressed Ad fiber protein. Furthermore, Michael S. I. [Michael S. I. et al. Gene Therapy 2:660-668 (1995)] described a new cell specificity in the Ad's binding obtained by genetic fusion of a peptide ligand to the carboxyl terminal of the Ad fiber protein. At the 3′ end of the coding region of the Ad5 fiber gene, a coding region of a physiological ligand, the terminal decapeptide of the gastrin releasing peptide (GRP), was introduced, resulting in the expression a fusion fiber-GRP protein product with a different specificity.

Other additional features which can be added to the vectors may ensure its safety and/or enhance its therapeutic efficacy. Such features include, for example, markers that can be used for selection, features that limit expression to particular cell types, such as promoters and regulatory elements that are specific for the desired cell type.

According to one embodiment of the invention, the therapeutic exogenous nucleotide sequence within the DNA construct of the invention is a sequence which either encodes a therapeutic exogenous protein or peptide product or is itself a therapeutic product or encodes a therapeutic RNA, or comprises a vector comprising exogenous DNA encoding a therapeutic exogenous protein or peptide product or a therapeutic RNA.

More specifically, the exogenous nucleotide sequence within the DNA construct of the invention is a sequence which encodes a therapeutic protein or peptide product which is not made or contained in said cell, or is a nucleotide sequence which encodes a therapeutic protein or peptide product which is made or contained in said cell in abnormally low amount, or is a nucleotide sequence which encodes a therapeutic protein or peptide product which is made or contained in said cell in defective form or is a nucleotide sequence which encodes a therapeutic protein or peptide product which is made or contained in said cell in physiologically abnormal or normal amount, or encodes a therapeutic RNA.

Preferably, the therapeutic protein or peptide product encoded by the exogenous nucleotide sequence is an enzyme, a receptor, a structural protein, a regulatory protein or a hormone which may be a naturally occurring or recombinant protein or peptide product or a modified protein or peptide.

Alternatively, the DNA constructs may comprise exogenous nucleotide sequences which are themselves therapeutic sequences. Such sequences do net necessarily encode a protein or peptide product, but act, for example, as regulatory elements which are not contained in the cell, are contained in the cell in defective form.

It is understood, that the constructs of the invention may be utilized in gene therapy in a manner known to those skilled in the art.

The host cell transfected with the DNA construct of the invention may be a mammalian cell.

In a second embodiment of the invention, the peptide or protein product encoded by the exogenous nucleotide sequence is a protein or peptide product capable of eliciting in an animal protective immunity against a specific antigen. The animal may be a human or a domesticated animal, preferably a bird.

When the domesticated animal is a bird, the antigen will preferably be a pathogen selected from Infectious Bursal Disease virus (IBDV), Newcastle Disease virus (NDV), Egg Drop Syndrome adenovirus (EDS), Infectious Bronchitis (IB), Marek Disease virus (MDV), Avian Influenza virus, fowl pox virus, chicken anemia virus (CAV), laryngo tracheitis virus, salmonella, coccidia or bacteria causing fowl cholera such as pasteurella or any other pathogen.

In a third aspect, the invention relates to a DNA construct for the expression of a protein or peptide product in a host cell, comprising an expression vector and at least one nucleotide sequence of the invention or functional equivalents and fragments thereof operably linked to the expression vector, the protein or peptide product being capable of eliciting in an animal protective immunity against HEV.

While with the DNA constructs described hereinbefore the transfection and/or expression vectors are comprised of the nucleotide sequence of the invention and the nucleotide sequence to be transferred into the host cell's genome is foreign, in the present DNA construct the vector, being an expression vector, is comprised of a foreign DNA sequence and the nucleotide sequence to be transfected is of HEV origin. Thus, the DNA construct disclosed in the third aspect of the invention is employed to transfect or infect a host cell with sequences encoded for antigens against HEV.

Within the same aspect, the preferred animal is a domesticated bird and the expression system is selected from the group consisting of fowlpox virus, vaccinia virus, Marek disease virus, baculovirus, bacteria, yeast and plant cells.

In a fourth aspect, the invention relates to a host cell transformed with a nucleotide sequence of the invention or transfected with any one of the DNA constructs of the invention. In one embodiment, the host cell is capable of expressing the therapeutic protein or peptide product encoded by the exogenous nucleotide sequence within the DNA construct, the protein or peptide product being capable of eliciting protective immunity against a specific antigen.

In a second embodiment, the host cell, transfected with the DNA construct of the invention, is capable of expressing a protein or peptide encoded by the exogenous nucleotide sequence within the DNA construct, which protein or peptide is capable of eliciting protective immunity against HEV.

The host cell may be a eukaryotic host cell, wherein said eukaryotic host cell is an insect cell, a plant cell, a mammalian cell, a bird cell, or a yeast cell, or a prokaryotic host cell such as a bacterial cell.

In yet a fifth aspect, the invention relates to a protein or peptide product expressed by the host cell of the invention, or by any other host cell transformed with the DNA sequence of the invention or transfected with the DNA construct of the invention.

The protein or peptide product may be a therapeutic protein or peptide product which is not made or contained in said cell, or is a therapeutic protein or peptide product which is made or contained in said cell in abnormally low amount, or is a therapeutic protein or peptide product which is made or contained in said cell in defective form or is a therapeutic protein or peptide product which is made or contained in said cell in physiologically abnormal amounts. Preferably, the protein or peptide product being an enzyme, a receptor, a structural protein, a regulatory protein or a hormone and may be a naturally occurring or recombinant protein or peptide product or a modified protein or peptide.

Alternatively, the protein or peptide product expressed by the host cell of the invention, or any other host cell as detailed above, may be such that is capable of eliciting in an animal protective immunity against a specific antigen, the animal being a human or a domesticated animal. Such peptides may also be employed as antigens in specific immunoassay tests (e.g. ELISA) or may be injected to produce antibodies for diagnostic purposes.

Further, the protein or peptide product may be a product encoded by the DNA sequence of the invention. In particular, the protein or peptide product may comprise a sequence substantially as shown in SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31 or functional equivalents and fragments thereof provided that when said fragment is comprised within said SEQ ID NO:23, it does not correspond to the segment corresponding to SEQ ID NO:6 described in PCT/IL98/00609, incorporated herein by reference, or to derivatives thereof This sequence corresponds to the segment within SEQ ID NO:22, starting from amino acid 63 to amino acid 454.

The above sequences encode the following HEV proteins: 52K, IIIa, CPI, CPII, pVI, EP, 100K, pVIII, the complete fiber, Iva2, POL, pTP and DBP, respectively. In this case, the protein or peptide will be capable of eliciting in an animal protective immunity against HEV and the animal is a domesticated bird. Such peptides may be employed as antigens in specific immunoassay tests (e.g. ELISA) or may be injected to produce antibodies for diagnostic purposes (e.g. for the specific identification of HEV or anti-HEV antibodies).

Yet, in another aspect of the invention, a process for the production of transformed or transfected host cells of the invention is disclosed. The process comprising the steps of (a) transforming a host cell with the DNA sequence of the invention or transfecting a host cell with a DNA construct of the invention; (b) isolating the transformed or transfected cells obtained by step (a); and (c) culturing the host cell isolated in step (b) under conditions in which propagation of said cells takes place. Such steps may be conducted by any suitable method known to the man of the art The process itself may be used for ex vivo transfection in gene therapy.

A process for the production of a protein or peptide, is also within the scope of the invention. The process comprises the steps of (a) transforming a host cell with the nucleotide sequence of the invention or transfecting a host cell with a DNA construct of the invention; (b) culturing the cells obtained in (a) under conditions in which an expression of the protein takes place; and (b) isolating the expressed protein or peptide from the cell culture or/and the culture supernatant. Evidently, the suitable means to performs such steps are well know to the man of ordinary skill in the art [ThioFusion Expression Systems Version 1.1 Invitrogen Corporation San Diego, Calif. (1995); Pichia Expression Kit, Version 1.8, Invitrogen Corporation San Diego, Calif. (1995); and Pitcovski J. et. al. Avian Disease 40:75-761 (1996)].

The invention further relates to a vaccine for immunizing a domesticated animal against at least one specific antigen (hereinafter referred to as the first vaccine) which vaccine comprises an effective amount of the DNA construct of the invention or the protein or peptide of the invention, optionally further comprising pharmaceutically acceptable carriers, diluents and additives.

The term ‘effective amount’ for purposes herein is that determined by such considerations as are known in the art. The amount must be sufficient to alter the transformed cell's function or sufficient to stimulate the immune system and confer immunity against the specific antigen and preferably to confer immunity to progeny of the treated animal, via maternal antibodies.

By the terms carriers, diluents, adjuvants and vehicles it is meant any inert, non-toxic solid or liquid filler, diluent, or encapsulating material, not reacting with the active ingredient of the invention.

Up to date, different vaccines employing Ad origin vectors have been described [Karen F., et al., Current Opinion in Genetics and Development 3:499-503 (1993); Mason B. B. et al. Virol. 177:462-461 (1990); Fields Virology Vol 2. Fileds B. N. et al Eds. Lippincott-Raven publication; Prevec L. et al. The J. of Infectious Diseases 161:27-30 (1990); Xiang Z. Q. Virol. 219:220-227 (1996); Callebaut P. et al. J. of General Virol. 77:309-313 (1996)].

In yet another embodiment, the invention relates to a vaccine for immunizing an animal against HEV (hereinafter referred to as the second vaccine) comprising an immunologically effective amount of any one of the nucleotide sequences of the invention or any of the DNA constructs of the invention or any protein of the invention encoded by the DNA sequences of the invention, and optionally further comprising pharmaceutically acceptable carriers, diluents and other suitable additives. The DNA constructs according to this embodiment are (as elaborated hereinbefore) those constructed from a HEV nucleotide sequence to be expressed, operably linked to a foreign expression vector being capable of expressing the HEV sequence. The encoded protein or peptide product will then act as HEV antigen against which an immune system may be elicited.

Both types of vaccines according to the invention may be provided in various forms such as lysates of the cells of the invention, as partially or completely purified proteins of the invention, as the DNA constructs of the invention and is preferably a sub-unit type vaccine, nevertheless, not limited thereto.

The present invention also concerns with pharmaceutical compositions comprising as active ingredient a therapeutically effective amount of a DNA construct according to the invention, or of a cell according to the invention, or of a therapeutic protein or peptide product according to the invention. The pharmaceutical composition of the invention may also further comprise any suitable additive such as pharmaceutically acceptable carriers, diluents, adjuvants and vehicles.

The vaccines or compositions of the invention are administered and dosed in accordance with good veterinary practice, taking into account the clinical condition of the individual animal, the site and method of administration, scheduling of administration, the animal's age, body weight, diet and other factors, well know to the veterinary practitioner. The doses may be single doses or multiple doses and the treatment may be effected at any age from day one.

In addition, the invention relates to a method of providing a therapeutic exogenous nucleotide sequence to an animal in need of such sequence by administering to the animal a therapeutically effective amount of the DNA construct of the invention.

Furthermore, the invention relates to a method of providing a therapeutic protein or peptide product, to a patient in need of such product, by administering to the patient therapeutically effective amount of the DNA construct of the invention or a therapeutically effective amount of the transfected cells of the invention [for example, as described in a Clinical Protocol in Human Gene Therapy 5:501-519 (1994)].

Any conventional method for as administering the products of the invention (e.g. the DNA sequences, the DNA constructs, or the protein or peptide product of the invention), such as tablets, suspensions, solutions, emulsions, capsules, powders, syrups, and the like may be used, as long as the biological activity of the therapeutic ingredient thereof is retained.

Administration may be oral, subcutaneous or parenteral, including intravenous, intramuscular, intraperitoneal and intranassal administration as well as intrathecal and infusion techniques. Nevertheless, most preferred methods are oral administration and injection. Following injection the DNA construct of the invention will circulate until it recognizes the host cell with the appropriate target specificity for infection.

In addition, the invention relates to a method of immunizing an animal against a specific antigen by administering to said animal an effective immunizing amount of the first vaccine according to the invention, wherein the animal is preferably a human or a domesticated animal.

Alternatively, the invention relates to a method of immunizing a domesticated animal against HEV by administering to the animal an effective immunizing amount of the second vaccine according to the invention, which case, the animal is preferably a bird.

The invention also relates to the use of the nucleotide sequences of the invention, or the DNA constructs of the invention or the protein and peptide products of the invention in the preparation of a vaccine or of a pharmaceutical composition.

Finally, the invention relates to an antibody, either monoclonal, polyclonal or recombinant antibodies, directed against any one of the DNA sequences or the protein or peptide products of the invention. The antibodies may be produced by standard antibody production technology well known to those skilled in the are, as described generally [Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor N.Y. (1988); Borrebaeck et al. Antibody Engineering-A Practical Guide, W.H Freeman and Co. (1992)]. In addition, antibody fragments may be prepared from the antibodies by methods known in the art and will include the Fab, F(ab′) 2 and Fv fragments. Evidently, such antibodies may be used in detecting the presence, in a biological sample, of the specific antigen against which they have been produced. Such methods are known to those skilled in the art and may include the ImmunoComb technology (to Orgenics).

The invention will now be described in an illustrative manner and it is to be understood that the terminology which will be used is intended to be in the nature of is the words of description rather than of limitation.

Obviously, many modifications and variations of the present invention are possible in light of the above teaching, it is therefore, to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described.

EXAMPLES

Example 1 Isolation of HEV

Turkeys were exposed to the virulent field strain of HEV. Five days later, birds were sacrificed, and the spleens were removed. A TCN solution (10 mM Tris-HCl pH 7.2, 10 mM CaCl 2 , 100 mM NaCl) twice the volume of the tissue was added to the spleens which were then ground and homogenized for 5 minutes (min.). The tissue homogenate was frozen and then thawed at −70° C. and at 37° C., respectively, for three times, followed by centrifugation at 8,000×g at 4° C., for 20 min. The supernatant was mixed with trichlorotrifluoroethane (1:3) and further centrifuged at 5,000×g at 4° C., for 30 min. The resulting supernatant was then collected and added on top of the following gradient: 12 ml of 46.2% (w/v) cesium chloride (CsCl) (density of 1.35 g/ml); 12 ml of 35% (w/v) CsCl (density of 1.24 g/ml); 6 ml of 1M sucrose. The gradient was centrifuged for 24 hours (hrs) at 85,000×g at 4° C., with SW28 rotor.

The virus was isolated from a white ring, formed between the two CsCl layers obtained after centrifugation. The virus band was collected, diluted in Tris EDTA (TE), and repelleted by centrifiugation at 96,000×g for 2 hrs. The pellet was collected, resuspended in distilled water and dialyzed against TE. The virus obtained was stored at −20° C.

Example 2

Isolation and Purification of HEV DNA

The virus obtained as described in Example 1, was incubated for 3 hrs in a solution containing 0.01M Tris, 0.01M NaCl, 0.01M EDTA, 0.5% SDS, and 50 mg/ml proteinase K. Following incubation, the DNA of HEV was electrophoresed on 0.8% agarose gel and was visualized by Ethidium Bromide, at the size of 26 kb.

The plasmids obtained were transformed into E. coli XL1-blue cells, and white colonies, that grew on Luria Bertani medium (LB) plates containing ampicillin (100 g/ml) and X-Gal (200 g/ml), were isolated.

Example 3

DNA Sequencing

HEV DNA was digested either by EcoRI or PstI restriction enzymes and the resulting restriction fragments were separated on agarose gel, purified and cloned into plasmid pBS SK(+) (Stratagene). Cloned DNA fragments which were longer than 3 kb, were digested by exonuclease III (Promega) to create a series of nested deletions. The initial sequence information was obtained by sequencing the cloned fragments, using two commercially available primers, corresponding to the 5′ and 3′ ends of the pBS multiple cloning site (universal primers, New England Biolabs). The sequencing was carried out using the Taq Dyeseoxy Terminator system and an ABI 373 automatic sequencing apparatus. Sequences of 350-500 bp were resolved in a typical sequencing run. Once the initial sequence information was obtained, the primer walking methodology was employed to complete the sequencing of each cloned fragment. Gaps between the separate contiges were bridged by PCR amplification of the viral DNA and a subsequent sequencing of the PCR products. When needed, as in the case of the genome terminus, the HEV genome was sequenced directly (i.e. without the need to clone the same into plasmids).

Sequence Assembly

The processing of the raw sequence data, in the form of ABI trace filed, and the assembly of the separated readings, first into contiges and then into complete genome, were done employing a sequence assembly program and other programs as detailed by Bonfield et al. [Bonfield J. K. et al. Nucleic Acid Res. 24:4992-4999 (1995)].

Both strands of the entire viral genome (referred to hereinafter as the rightward and leftward strands) were sequenced and each nucleotide was determined at least three times.

Example 4

Sequence Analysis

Sequence analysis was performed by means of Wisconsin Package Version 9.1 [The Univrsity of Wisconsin Genetics Computer Group software package (GCG), Madison, Wis.].

At first stage, a search for homology with sequences of other members of the Ad family was first conducted using the BLAST program, followed by a search, to obtain a higher degree of accuracy, conducted by means of the FASTA or TFASTA programs. Multiple sequence alignments were performed either by Pileup program [GCG package] or by ClustalX [Higgins D. G. & Sharp P. M. Gene 73:237-244 (1988)].

The coding regions in the HEV genome were identified by comparison of the sequence obtained with sequences, pooled from the GenBank, of human Ad 2 (Accession No. J01917), human Ad 12 (Accession No. U40839), human Ad's 40 (Accession No. L19443), egg drop syndrome (EDS, Accession No. Y09598), CELO (Accession No. (U46933), ovine Ad (Accession Nos U18755, U40837, U31557, U40839) and canine Ad (Accession No. U55001).

Identification of the ORF of the HEV DNA Sequence

The complete sequence and genome organization of HEV was established. The genome map is presented in SEQ ID NO. 1 and in FIG. 1 in which identified regions are indicated.

The Genome length of HEV consists of 26269 bp, thus being the smallest genome isolated and characterized among the Ad family members [Jucker M. T. et al. (1996) ibid]. The overall G+C content in the genome is 34.93%. The inverted terminal repeats (ITR) are 39 bp long. As regards to these parameters, (size of the genome, G+C content, ITR) HEV resembles ovine Ad, consisting of 29544 bp long genome, 33.6% G+C content and 46 bp in the inverted terminal repeats.

The location of HEV genes as found by the inventors is summarized in Table 1.

TABLE 1
Summary of ORF locations and features in the HEV genome
(as compared to ovine Ad)
	SEQ ID NO			No. of
Protein	(na and aa)*	ATG	Stop	residues	Mol. wt
L1
52K	NO:2; NO:3	8569	9468	300	33800
IIIa	NO:4; NO:5	9461	10975	505	50293
L2
PB	NO:6; NO:7	11000	12343	448	50903
CP I	NO:8; NO:9	12346	12705	120	11134
CP II	NO:10; NO:11	12711	12884	58	6111
L3
pVI	NO:12; NO:13	12905	13597	231	24947
Hexon	NO:14; NO:15	13609	16326	906	101089
EP	NO:16; NO:17	16331	16972	214	25008
L4
100K	NO:18; NO:19	18184	20223	680	78283
pVIII	NO:20; NO:21	20768	21367	200	21769
E3		21213	22115	300
L5
Fiber	NO:22; NO:23	22518	23879	454	48770
IVa2	NO:24; NO:25	3436	2336	367	41802
E2B
POL	NO:26; NO:27	6767	3432	1112	129160
pTP	NO:28; NO:29	8557	6767	597	70582
E2A
DBP	NO:30; NO:31	18012	16975	346	38910
*SEQ ID NO for the nucleotide sequence (na) and the amino acid sequence (aa)

Comparison of these sequences with known and new members of the Ad family (Table 2) enables to determine the degree of homology between such members. The comparison shown in Table 2 was obtained using the Gap program for global comparison which is part of the GCG version 9 software package. The gap opening penalty parameter of Gap was set to 6 and its gap extension penalty parameter to 2. The sequences of other Ad's were pooled from the GenBank (accession Nos. as described hereinbefore). Abbreviations are POL for DNA polymerase, pTP for precursor terminal protein, DBP for DNA binding protein and EP for endoproteinase. The percent of amino acid sequence identity (%id) and similarity (%s) is presented in table which exhibit the uniqueness of the HEV genome.

TABLE 2
Comparison of the amino acid sequences of HEV proteins,
with Ad's of different species
Virus	Ad2	EDS	Ovine	CELO
Protein	% id	% s	% id	% s	% id	% s	% id	% s
52K	23.4	35.5	27.1	34.9	25.6	34.4	28.7	40.4
IIIa	31.2	40.9	34.2	43.7	32.3	44.3	30.8	41.2
penton	48.6	58.1	52.9	61.4	52.6	61.4	49.7	59.3
PVI	29.9	38.4	37.3	45.0	37.3	45.6	41.0	49.7
hexon	52.8	59.4	53.	62.3	55.9	63.3	54.7	62.0
EP	44.2	53.8	44.8	55.7	41.0	52.5	50.7	59.5
100K	36.1	45.3	38.4	47.5	36.6	47.4	38.6	48.8
PVIII	23.1	33.7	30.1	37.7	29.3	35.4	24.8	33.4
fiber	25.7	33.6	30.1	37.6	29.3	35.4	24.8	33.4
IVa2	33.3	44.3	35.3	46.5	36.9	46.2	36.7	46.9
POL	41.1	52.9	43.7	54.9	44.8	54.5	42.0	53.7
pTP	34.3	44.8	37.4	45.8	34.5	46.6	35.9	47.7
DBP	28.4	40.5	28.4	40.5	36.0	43.7	34.4	43.9

Characterization of the HEV Genome Regions in Comparison to Other Members of the Ad Family

In general, a genome is characterized by two major areas, the early region and the Late region, according to the stage of translation thereof. Each region comprises sequences which, inter alia, encode for different products, as detailed, for example, in Table 1.

In an attempt to identify the content of each region, the inventors deduced the following information.

Early Region (E)

E1—In general, in human Ad's (as deduced from human Ad 5), the E1 region comprises E1A and E1B. E1A encodes two major proteins whereas E1B encodes two major proteins and later in infection three smaller proteins. E1A transactivates expression of E3, E4 and partially E2 [Shenk, T. & Flint, J. Adv. Cancer Res. 57:47-85 (1991)] and induced DNA synthesis and cell transformation [Bayley, S. T. & Myniryk, J. S. J. Oncol. 5:425-444 (1994)]. In addition, this region is plays a role in the stimulation of specific human gene expression of infected cells.

No functional homologous region to this E1 was identified in the corresponding location in the HEV genome. Some ORF's were identified in sites in which E1 genes are located in other Ad's genomes, however, these sequences are not similar to any E1 sequence or to unassigned ORF's in other aviadenoviruses.

E2—E2 region encompass E2A and E2B regions and is located in the genome from 18013 to 16976 and 8557 to 3432, respectively. The E2A region consists of a gene which encodes the DNA binding protein (DBP). The DBP is divided, in Ad2, into two clusters. The C-terminal cluster is involved in binding to the viral DNA and activating replication of the major late promoter. High identity levels were found in this cluster among the C-terminus of DBP's from various Ad's. DBP is located between the EP and 100K genes, on the complementary strand. The DBP size, in HEV, is 345 amino acids long which is smaller than in other Ad's (382, 387, 441 and 539 in Ova, EDS, CELO and Ad2, respectively). The sequence VFQCCNP of HEV DBP (amino acids 255 to 261, SEQ ID NO:32) was found to be conserved in Ad2, ES and Ova. The Ova has a charged motif, KKRK (amino acids 11 to 14, SEQ ID NO:41) which is used for nuclear localization and it seems the HEV DBP has a similar motif KKNK (amino acids 35 to 38, SEQ ID NO:42) which may have the same function. A motif comprising Leu 515, Pro 516 and Pro 526, that serves in the cooperative binding of the subunits is partially conserved in HEV and correspond to residues Leu 316, Pro 317, and Pro 323. this motif was completely conserved in EDS and is missing from CELO.

The E2B region contains a sequence which encodes essentially two functional proteins, a DNA Polymerase (POL) and a pre-terminal (pTP) protein.

The pTP primes the Ad DNA replication [Salas, M. Science 149:1108 (1965); Smart, J. E. & Stillman, B. W. J. Biol. Chem. 25:13499-13506 (1982); Stillman, B. W. et al. Cell 23:497-508 (1981)]. This protein is processed at two sites by viral protease to a mature terminal protein (TP), which takes place through two cleavages by viral endoproteinase and was shown to be conserved among the different serotypes of the Ad family [Webster A. et al. J. Virol. 71:6381-6389 (1997)]. in HEV, pTP consists of 597 residues. Two cleavage sites were identified, at residues 169 and 297, which is similar to the terminal protein of EDS (consisting of 581 amino acids and is cleaved at residues 156 and 265). A sequence similar to that reported as a nuclear localization signal in Ad2 (RLPVRRRRRRVP, residues 380-391, SEQ ID NO:33) and in EDS (TLPARTRRTRRP, residues 308-319, SEQ ID NO:34) was identified in HEV (SLPLIRRIRRPP, residues 341-352, SEQ ID NO:35).

E3—An ORE of 900 bp lays between the regions coding for HEV pIIIa and the fiber protein (nucleic acids 21214 to 22113in SEQ ID NO:1). This ORF bares only little similarity to one of the gene products of the predicted E3 of Ova [Vrati S. et al. (1996) ibid.]. E3 has been mapped to this region in most Ad's. Two short additional ORF's are found near this ORF, both have no similarity to any published Ad sequence (data not shown). One of the E3 products, E3gp19K, binds to MHC class I antigens in the endoplasmic reticulum and blocks the cytolysis of viral infected cells. Such an interaction is species specific [Hermiston, T. W. et. al. Virology 238:145-156 (1993)]. The size of E3 varies from 2.5 kb in human Ad to 1.0-1.5 kb in canine Ad [Dragulev, B. P. et. al., Virology 183:298-305 (1991)] and only 0.5 kb in mouse Ad [Raviprakash, K. S. et. al. L. Virol. 63:5455-5458 (1989)].

E4—In general, E4 region modulates viral gene expression and DNA replication and inhibits host cell protein synthesis by interacting with host cell systems [Leppard, K. N. et al. J. Gen. Virol. 78:2131-2138 (1997)]. E4 in human Ad has a sequence similar to that in bovine Ad however, a lower degree of similarity to other Ad's such as ovine Ad [Vrati, S. et. al. (1996) ibid.] and murine Ad [Ball, A. O. et al., Virology 180:257-265 (1991)] and a possible similarity to E4 in CELO [Weiss, R. S. et al. J. Virol. 71:1857-1770 (1997)]. Nevertheless, no homologous sequence to E4 was found in HEV genome.

Delayed Early Genes

In general, a pIX gene, known from other members of the Ad family to be located in this region, strengthen hexon-hexon interactions [Boulanger, P. et al. J. Gen. Virol. 44:783-800 (1979); Van Oostrum, J. & Burnet, R. M. J. Virol. 56:439-446 (1985)] and has transcriptional properties, such as stimulating the major late promotor [Lutz, P. & Kedinger, C. J. Virol. 70:1396-1405 (1996)].

No sequences homologous to pIX were found in any of the avian Ad's or in ovine Ad.

The IVa2 gene, also located in this region, was recently identified as a component which contributes to the activation of the major late promoter [Lutz, P. & Kedinger, C. (1996) ibid.], is shorter in HEV compared to other Ad's.

Late Region (L) Proteins

The identification of the genes in the ORF's of the late region were deduced by comparison to the genomes of EDS and adenovirus 2 (Ad2), pooled from the GenBank (accession numbers Y09598 and J01917, respectively). The comparison is depicted in FIG. 1 .

L1—The L1 region comprises the genes which encodes the 52K protein and the IIIa protein. The IIIa protein is known to bridge between the hexon and the core proteins.

L2—The L2 region comprises the genes encoding the structural protein, penton base (PB) and core protein I and core protein II (CP I and CP II, repetitively).

The penton base is bound to hexon protein at each vertex of the icosahedral structure. It is the base for the fiber, and together they constitute the penton. In most human Ad's, a tripeptide sequence, Arg/Gly/Asp (RGD) was identified and reported ad the binding site of the virus to cellular integrin, causing thereby endocytosis of the virus by the cell [Mathias, P et al. J. Virol. 68:6811-6814 (1994)]. A corresponding sequence was not identified in HEV, as in other avian Ad's and ovine Ad. The fact that no RGD sequence was found on the penton base indicates that the penetration of this Ad differs from that described for human Ad.

The core protein I and II (CP I and CP II) genes are located between the coding genes of PB and pVI proteins. CP I was found to be rich in arginine (17%), proline (11%) and alanine (13%). The molecular weights of CPI and CPII (11 kDa and 6 kDa, respectively) are smaller than the corresponding proteins in human Ad2 (48 kDa and 18 kDa) but are similar in size to the corresponding proteins in CELO (20 kDa, 12 kDa and 9.5 kDa). The core of human Ad's are slightly different, consisting of four proteins, pV, pVII mu and teiminal protein wherein PV, pVII, seems to be the counterparts of CPI and CPII.

L3—The L3 region contains genes encoding the pVI protein, the hexon protein and the endoproteinase (EP).

The pVI nucleotide sequence of HEV is substantially of the same size as in other avian Ad's. Two cleavage sites for the viral endoporteinase wee identified on both ends of the protein. The sequence LRGGK (residues 24 to 28, SEQ ID NO:36) in the N-terminal resembles a conserved cleavage site [Freimuth, P. & Andreson, C. W. Virology 193:348-355 (1993)]. Cleavage at the C-terminal of the pVI protein, gave rise to a peptide of 11 amino acids (FIG. 2 ). Sequence alignment of this peptide exhibits partial homology to C-terminals of several other pVI proteins. This 11 amino acid peptide was shown to act as a cofactor of the viral protease [Mangel, W. F. et. al. J. Virol. 68:6811-6814 (1993)] by forming a disulfide bond with the protease via a conserved cysteine residue (FIG. 2 ). The conserved sequence of this peptide was proposed by Vrati et al. [Vrati, S. et. al. (1996) ibid.], and is GXXXXXRRY/RCF/Y (SEQ ID NO:37, in which Y/R and F/Y are denoted by X).

The hexon protein, being a monomer of the major outer capsid is important in the determination of the capsid diameter. The HEV hexon protein (97 kDa) was found to be similar in size to other Avian Ad's.

The gene encoding the HEV endoproteinase (EP) aligned with the amino Acid sequences of other Ad's (CELO, EDS, Ad12 and Ad2) is depicted in FIG. 3 which shows that the HEV EP has 9 additional amino acid residues at the C-termiinal thereof When comparing with the active site of the viral EP of Ad2 which comprises amino acids His54, Glu71 and Cys122, the Glu71 residue is replaced in other Ad's by a similar amino acid, the aspartic acid (FIG. 3 ). In addition, these three amino acids are surrounded by a highly conserved region which probably forms the active cavity. In the process of the viral maturation, the EP cleaves several structural proteins. The cleavage site comprising the sequence (MLI)XGXG or (MLI)XGGX (SEQ ID NOS:38 and 39, respectively) as determined in other Ad's, was also identified in HEV EP, in a similar location. Table 3 shows that EP is one of the most conserved proteins of the Ad's

L4—the L4 region comprises the genes encoding for the 100K protein and the pVIII protein. it seems the 100K protein is required for assembly of the hexon. Possibly, the 100K protein is required also for translation of the Viral RNA.

L5—the L5 region comprises the gene encoding another structural protein, the fiber protein consisting of a globular head, a shaft and a tail. The fiber protein plays an important role in the first attachment of the virus to the cell receptor, which is specific and with high affinity [Mei Y. F. & Wadell P. C. EMBO J. 11:751:760 (1992)]. All the elements described for the fiber protein of other Ad's were identified in HEV fiber ( FIGS. 5A-1 , 5 A- 2 and 5 A- 3 ).

Undefined ORF's

Eight ORF's encoding for putative polypeptides consisting of more than 100 amino acids were detected in the HEV genome. The relative location of these ORF's was found to correlate with the E1A, E1B and E4 regions identified in other Ad's. However, no functional homology was detected between these polypeptides and products encoded by these early regions, in other Ad's. Additional unidentified ORF's coding for shorter polypeptides, comprising only 50-100 amino acids, were found scattered throughout the HEV genome, 16 on one strand and 21 on the other strand.

Control Regions

Major late promoter—A TATA box for the major late promoter was identified starting at nucleotide 5384 (TTATATT). The ATG codon (+1) was found 30 nucleotides upstream. The CAAT sequence is located between nucleotides −81 to −78.

Stop codons—for several genes, as additional stop codon is present within 20 nucleotides downstream from the recognized stop codon (FIG. 6 ). It is possible that it is suggested that such additional stop codons ensure the accurate termination of translation.

Inverted Terminal Repeats—although relatively short, (39 bp), HEV ITR's include two conserved motifs identified in other Ad's (FIG. 4 ). Within the more conserved region, between nucleotides 9 to 18, only an arginine residue, at position 12, is missing from HEV ( FIG. 4 a ). In human Ad, this Arg residue was found to be a part of a TAAT region which is involved in the initiation of DNA replication [Rawlins, D. R. et. al. Ann. Rev. Biochem. 60:39-71 (1984); Stillman, B. W. et. al. Cell 23:497-508 (1982)]. It has been suggested [Chen M. et al. J. Biol. Chem. 265: 18634-18642 (1990)] that a complex of two viral proteins (pTP and POL) bind to this sequence.

In addition, a GGGNGG region (nucleotides 26 to 31, SEQ ID NO:40), consisting part of a domain referred to in human Ad's as domain B (nucleotides 19 to 39) may correspond to the sequence reported as binding the SP1 transcription factor. An additional domain, referred to in human Ad's as domain C consists of nucleotides 40 to 51. Cellular factors bind to domains B and C to enhance the efficiency of the of the initiation of DNA replication. It seems that HEV consists of domain B however is missing domain C ( FIG. 4 b ).

Table 2 exhibits ORF's of HEV, to which, when compared to other published Ad sequences, no homologues were found.

TABLE 2
ORF to which no homologous sequences were found in comparison to any
other published adenovirus sequence
	START	STOP	No. of residues	Mol. wt
ORF1	399	1139	246	27327
ORF2	616	918	100	11220
ORF3	1220	1951	243	26953
ORF4	1497	1871	124	14495
ORF5	4477	4974	166
ORF6	17178	17537	120	14004
ORF7	24509	24823	104
ORF8	24847	25164	106	11902

Overlap—very short overlaps appear in the major genes. Only four of the unassigned ORF's seem to overlap with identified genes.

Example 6

Identification of Non-essential Regions in the DNA of HEV and Insertion of Foreign DNA Thereto

Engineered virus vectors are developed, inter alia, for purposes of recombinant vaccination by way of expression of antigens from one or more pathogens, or for gene therapy (e.g. for the production of therapeutic proteins or peptides). In order to construct viral vectors, non-essential regions in the DNA sequence of the virus to be engineered must be identified. These sequences are then removed, to increase the capacity of the vector and enable the insertion of desired foreign DNA's.

Non-essential regions in the DNA sequence of HEV may be determined experimentally by adding foreign marker genes at particular sites and identification of viral replication and protein expression. In general, the genome of Ad's, as studied from the genome of Ad5, contains two regions frequently used for insertion of foreign DNA [Moss, B. et al. Vaccine 6:161-163 (1988)]. These are the early region 3 (E3), a lot of which can be deleted without interfering with the ability of the virus to replicate in cultured cells, and the early region 1 (E1), which is essential for viral replication in most human cell lines.

For high level expression of proteins of peptides, the most suitable vectors may be those containing substitutions in E1, with transcription driven by strong promoters. The construction of vectors of this type, in which transcription of inserts is under the control of an extra copy of the Ad major late promoter, has already been described.

Nonetheless, for use as vaccines, substitution of E3 sequences with genes encoding foreign proteins or peptide products such as specific antigens is likely to be the most promising approach since this results in vectors that can replicate both in most human cells in culture and in animals. Deletion of E3 sequences results in vectors with capacity of 4-5 kb inserts. Inserts in E3, in the left to right (E3 parallel) orientation are generally expressed as transcripts originating either from the E3 promoter, or from the major late promoter. Amounts of protein produced in cells infected with such vectors vary depending on the inserted gene but are almost invariably adequate to induce a good immune response in appropriate animals.

The construction of engineered Ad vectors, comprising a foreign gene, may be performed using standard techniques [Molecular Cloning, a laboratory manual, 2nd Eds., Sambrook, J.; Fritsch, E. F.; Maniatis, T. Cold Spring Harbor Laboratory Press (1989)]. For example, the foreign gene to be expressed is first inserted into a subgenomic viral DNA segment propagated in a suitable plasmid, for example, a bacterial plasmid. The resulting construct is ‘rescued’ into fill length infectious viral DNA by cotransfection into tissue culture cells with either a second viral DNA segment or a second plasmid, containing the rest of the viral genome plus the overlapping sequences required for homologous recombination. Identification of the desired viral recombinant is by plaque isolation and analysis of viral DNA structure by restriction and gel electrophoresis.

42 1 26270 DNA hemorrhagic enteritis virus misc_feature (25290)..(25290) N=Unknown 1caatcaatat atataccgca tgcttgggag gggatttcgg cttgaaaagt gattttcttt 60ttactgtact tttctttttg ttttctacat gtttcatttt ttgttttctg gttattttac 120gtttaaattg tctaattgct tttaaaaagt tgatttgttc gttttctgtt tgaattgtgg 180gcggttaaat tctgtgtcga tcttggtcat gtgtttcgga ttggcgctta aagcgctttc 240atttcccgta aacggtgttg taattttcga gtcatcacaa ctgaccttgt cgtccgtgcg 300gggtaagacc atggagtttc ccttttctgt tactcctgat agccctaagt ggccagtgaa 360gcgaaaatgg gaagggatag aatgtggcta tataaagtat gggatacggg ctggagaata 420ttctccggct ggtccaaggg ctgtgcgtta ccctcttcca ttttgatgaa atagatagag 480agttggaatt tgattctcat gctttgaggt ctgttggaca aactgctgtt ttttcgcctg 540gagatttttc gtcagcattt ttcaaaaatc cttttgttat tgaattgcaa agtggtagat 600tgcttgctgg tggagatgtc agatatgagt ctcttaccgc agaccacagg attgacattg 660cagttgccat cagtgacgat ggaggactta cctggccaag gaagtcgttc gtgttacagt 720ctaagccgga tgtcgagaat ggaatgttca tggatggatg tattttggag gctccttcgg 780gagttgtgca cttgtttgcc gtgtactttg aaagtgcaaa ttataagtcc gttgtggacc 840ctgaatatga ctttgtgcac gtcacgtctg aggatggcgg gatcacttgg agtgatatta 900agtctctcaa gagcttgaag acgtcaaatg aagattattt ttttcaatgt ggcggcaatg 960gcttagtaat gtcaaatgga acactggttg ttccttgtgt ttcgtggaag gatggatttc 1020cacaatatag cactattatt tattcaacta atggagttga ctggacaaga cctcttatac 1080taatactatc atcaatgact gtgtggactg tcaagtgtct gaaattgatg gagatttagt 1140tagattagga agaaggccta ttacttattc tcctacaagt cctatagata atactttacg 1200attttttatt tcttctgata tgggcacaac ctgggaaacg cttcctgggg atagcacttt 1260aaaagtttgg cttggcgttt catgttcttt agttaacgta cacacagttg ataatgtgaa 1320tgtgttatta aatactgcaa tgtattatcg tgatgataat gacaaggcgt tgggtcttca 1380aatgtttttg tatccacatg ctcagtggag gcctgttgga attgttgcta attctgtttt 1440gactcggggt aatattgtgc aatctacgtc ttttggtaaa ttatttgttt tttctgatgt 1500ttcagttaat cagggtagcg ctttgtcact ttatgatatt agtaggtatt tttcatgtgt 1560atctactttg cctggatata atgttaattt gggttttcct gttactatcg aaggttctgc 1620gaaacttcca tcaactagcc cgctcaagtt taggtatgat gggagatctt actggattgg 1680gggatttctg gaacctagaa caggtggcac tttttctact tctcaacaag ttatgtttgc 1740aattagaatt ggctggccaa tttttaatga tggtaatatt gttgcttttg gacaaacttc 1800caatggtcaa ttgtatcctt ttttatttaa atatgaattt ttgtatggcg aattattttt 1860ttcatgttta gatacgtcta agttaactcc gtcactagct gcttgtactt cattatactt 1920tcctgagagt agaattggac tattttgtta atttctttca gaatgccttt cttctactta 1980gttggtgctg gcagtgcata ttgtgagcgt tgtaaagaag tttgtgaaaa gagaaggaag 2040aggtcaacaa caagaactac aagaactaaa agatctaaac cttctcacct tcaatatgtg 2100cgttactatc ctggcacagt ggtttcctgt tggctgggat ggtactgata agcctgttac 2160tgtcactaga attcctgact actggacgta cgacagagct gtttctagtc gtcaatctaa 2220tactgtggtt cctgttaata cttctggaga aaatccaact gtggttgctg tgccgagact 2280tttgcgaaaa aggaaagctt cagatatgtg aaaagtttga ataaaaattt tattaaattt 2340catcatacat gcgtttttgt ttttctttca agtacctttt cttatttttt aatttacata 2400agtgaatgtt acagatttta tttacagagt ctagaagtaa actttgcata tctaaacata 2460aaggtataat acttttccct ccctcgagta ttgctgacca ttgaaatgat tcccttgttg 2520gacaagtatt aaacattaca aaagaatatt tcggattttt ttcagataca atactgttta 2580atagtatcgt tcgtacgtta gagtccattc ctcctgatcg attatgtaca aaattagata 2640attgtagtgg ttggtttttt gtgcttaaaa tgtgtaattt ggcttgagtc ttcaagtcca 2700taatattatt accgtttcct gataacggat ttacattatg taaaacaacg aacatataga 2760atgcatgacc atatctacta gatagctttg atggtaaact acaatataat gggcttatat 2820ttggtttctg tattaatttc tgcatacatt catctagaat tacgcatact ggcccttttt 2880tggtatgaat attaaagata gaattttcgt tgttaacatc tagattctct ggggtaatga 2940catcatcaaa agcacattct agaaaattaa tggtgaatac ctttgtggtt gggtaaaggg 3000tattatcttg agcagaataa tttccttctt gtagttgagt tttccataat attacttcgt 3060catatgaaat agttcctttt gtaggagtta tgaagatgat cgtttctggc attggttgta 3120tttttttgaa tcccagtaaa tttctaatta attggctttt cccgcaacct gtaggtccga 3180ttactaaact aataagtgga tcagagttca tattgataga aggcagttca gagtttttag 3240ttagatacat cttgttaatg ttgttaattt cgttgtattt cctcatattg cttctcaagt 3300ctatattaga gttatagctt tcaaattctt taaatggcgg caaatcacat cctggtagta 3360tgcttgagtt aatactgtct acagcagttt tccattgtcc aactacatcg tagaattctt 3420caacttcatt attcatcttc gaattcctgt agtaagatag gatttgttat tctggggttt 3480gggtggtaga gatcataagg aattaaagtg tttgtcttta gaaagtaaag ggttggatca 3540ttccacggcc gtagttcgcg tattaattgt atttcatgaa cagaaaaggg ggagaatttt 3600ccgtaagact tacataacgt tcttttcaaa gcagttcttt ctgtttggaa gatctcacca 3660gattttaatt ctgaagtgtg gtagttgaag cattcttcca ggatttcaaa agttatgtct 3720gcggtacagt gaccctttgc tcgtaatttt cctgtttttg aatttttgca ggtggtacac 3780gtgatttctt taagggcgta cagctttgga gcgagaaaaa tactttttga agaatatgca 3840ggtgaattac acaaattaca ccaagtttca cattctactg cccaaacaat tgaaggtttt 3900ttaggatcaa atgttagttg tgaatttttg tttttaattc tatgttgtcc atattttaac 3960atattttgat gtcctttttc tgtgagaaat agactgtctg tgtcaccgta gattgattta 4020atggttttga attgaactgg tatactatct tcatcactgt ataggatttc gcgccactcg 4080ctcataaagg ctcttgtcca cgctaatacg aagcttgcta gttgagttgg atatcttttg 4140tttgtcggat gtaaattcgt tgattttaac atgtaaatag tgaggttttc tgatgtgacg 4200tctaagatgt tgaatggttt gtaggttccc acgtgagcag tcgaagttga agtttgtgag 4260gctgggcttt catccataaa ttctaaactg ttaaacggac tggtaagctc atcatccagt 4320tctaaatttc tctctgcact gtcagttcgg ttttttaagg taaatgttaa attttctata 4380ctaacatatg gaaggttgta agttggtaga gtagtaatgc tgtcaatggt tagttgtttg 4440ttcaataatt gatttcttat tttaggattt tcttgaatgt ggttttcgaa aattgtaatg 4500tcattgcttt ctcttgtagc aaaactaccg tatagtgcat tacttaggag tttgctgatt 4560gctcttttaa caggattttt ttcaacggtg gctttttctt ttgctagaat atttttagta 4620acatattcta agcagcaagt gttccacgtt gggaatactg tgtttagctt gtggggaata 4680atgtttactt tccatcctct attatggagt gtgataatat caattgaggt aacaatttca 4740tttttcagta tttcgttcgt ccaacataat ttgccgcttt gtcttgaaca taatggcggt 4800agaggatcaa gatgttctgg aggtggcggg atggcgtcaa tcattactac cattggcttt 4860atatcagtaa aatatgatag ttttgtttta gtttttttta atttttcgtt taactttgta 4920atttcattgt ttctttcttt ctctcctatt ggaaatccgt atggcattgg atgagttaga 4980gcactagcat acatgccgca aatatcgtaa acaaaaattt tttctttgaa tattcctaag 5040tgtgttgggt aacaacgtcc acctctaacg gactgacgga tgaaactgta catttcatca 5100gatggtgcta ctatgtctgg cagggtggag gcttttgttc cattttgttt gaagtgtaac 5160tgtctaaaaa tggcgtgaga attagatgaa atagtcggtc ttttaaaaat attaaaattg 5220cattttaggt ttagttcctc tttgatgaat gtatcgaaag tatttaggag ggtttttgtt 5280agttcttccg ttattttaac gtctttaata caatagtcga ttaattcttt aacaatgtca 5340tatttgactt cctttttttc taaccaaatt tctttttgtt cgttatattc ttctttgcta 5400gaccaatatt tttctgatgg aaacgagtct gagtctgtag ataatgaatt ggttgacatg 5460tactcattta tagctttgaa cggacaacaa cctttgtgaa tttttaagtt gtatgctttg 5520gccgcttctt ttagagaagt gtgtgttatt tggaaagtat ctctgaccat agacttgata 5580tataaatttt ttatactttc tggatgtgga attccttctt taatctgtga aagtatttca 5640cttttattat tttttttttc ttctttagct acatagtagt ctgggttggg gaatttaatt 5700gtaatatcgt taaaaagtat tctgccttgt cttggcataa aatttctttc aactgtgaaa 5760aggggagtga tgtctaggtt ttcgtcttga aggatttgtg ttgctaatag aatttcatcg 5820aatgactgaa tgttgtgtcc gatgatgtaa aattctatga aaattggttt aactttcaga 5880ctcattatga ggttagtatg ttcacttaaa ttgatatttg taattgattc taaattttgt 5940gtttgagcga aatcttcgag aatttctcgg ttttctggtg ttaaaattaa ttttgtgaag 6000taatctgtga gtgcaaatag gatgttattt cttaattttc tgaactccgt actaatgaag 6060tttttaactt tagaaaacca gaagtacgta gaattttttg tttgaataga ttttattttg 6120gctatttctg ttttacagat attgataaga caatcatctc cgaaaattga aaagcatagt 6180agaagaggat ttaataaaac tccagatgtt tcagctagtg taaatgtttc tatatcgtag 6240ataaggaaga gtttttttgt gttttcattt tctcctatag gttgaaatgg tatagtttcc 6300cagaattttc ttgtgtcatt gtctacgctg ttgtagtagt aagtagatct taagtcattg 6360caggtatgaa ttgcggagta gattctacca catgagctgc atttttgttg ttgagtccat 6420gattttatcc agcataattt tgacttatca tatatgaagt ctaattcaat atattcatgc 6480gatgttttga attcattttg gatcataaca catccagtta attttttcca gatagttatt 6540gatttgaccg gtaagttttt tacattttca atatcgctgt ttgtgggctt gtatttacta 6600caaatgagga tatatgattg tgcatctttt actttttttg caagtttttt acaattaaat 6660aaattatatg tccaagctat ttctattata gtggctttta tgtctttttt acatatttgc 6720aacctgtatg gaatttcgtt ttgagtggta taaatgtatt tgctcatctt ggttgtctta 6780atcttctcat ttcttgtgtt cttacagccg aagcattgtt aattatttgt ctgtttgtag 6840caattgttac aagaccaata ggcttgattt taaaggaaag agttactgtg ttgattaaat 6900cttcattaag tctagcttgg tttagaaggt cgtttggatc tccagattct ggtctgtgtg 6960atagggatgt aagtaggtct tcttgttctg cttgagtttc aatactatct ggccttctgt 7020cacatattat tagaaggtca ttgatgattg ttctaaatat tcttaaaaat ggagaaatgt 7080ttgtgttagt ccaaactctg tgaaggttaa catttccttc atgatctcgt ccagtcatta 7140tgacttggac atattgcata tttacatatc ttctgaaaat aacgtttaag tttaatagag 7200catgataata aaatagtgta ctactgatgt gttcagctat gaaaaagtaa aatatgaatc 7260ttcttataaa ctctctagtt acccttcctt gttcatttgc tctctgtaaa agattgtaaa 7320ataactgacc gaaagaaaaa atttcatgtt ctcttgcagg ttctgttaat tctgctctta 7380gttcattaag gatattttga aaaattcgaa ggatttcatt gcctaattct tcttccattt 7440cttcttcttc aggtccggca cttggacctt ctcctgcatc ttcctcttcc acaggacttg 7500gtgggggtct tcttattctt ctgattaaag gtaagctgtc tataaacctc gaaactatct 7560gtcctctatt tcttctcatt tgttcagtta tggctctttg attttctctc tgtcttaaaa 7620atggtaagtc tgttctagtg ccacttctta atcttgcacc accttttaga tttgttttcc 7680actctttgaa aggaaatatc attgaattta atgctgtgat atatgataca gcgtagttta 7740tatccatttc aggctgccat ttgtcgtatt ctgttttgag gatttgaagc caattatcct 7800tcaacggtac gtatattttt tcatctttaa attgccaatt ataaataaat tttgcaagta 7860gttctaaagt gtagttaatg cactttaatg tgttaatatc tttttcagtt tcatattgat 7920acctttgatt gaaagcaaaa cttttggagt taataaaatc tcttaaattt gttacgtaca 7980ttttatttat ttcattttgg atatttatat tttcgtaagc ttctggttgt aatgtaacac 8040ctgttccttg catgttactt ctagattcaa tgtcagctct aattctatta ataagaattt 8100cttcttgaaa ctggtttaag ttatcttcgt agttttctgg aatggtgcgc acattacttg 8160tgttaattgt atatgaacaa tcacttagaa tagaccaaaa atttctaggt cttcttgtag 8220tagattgatc agcttcatag tttaattttg tatatgttct gctttcaaaa cggtagttat 8280tgttcacatt gaatagataa gcgtagccaa tcagcaaatg aggaggcggc aatccgttta 8340aaggaggatt taaaacggca gggccttgtg gtgctaaatt ttctaacatt aaaatctgat 8400agttaaagaa tgtactgcac catcttaatc caggaatact tcttacaaac atagggatgt 8460tgacttgatc tgtgaattgt gttaaatgca taaatctgat ggtgtcttct tcttggttag 8520ttaggcgggc ataattgtta atctgaaaga aagacatttg tctttcagat gaataccatt 8580atgaaagcaa tgcaaacaga tagaggtaga ctagaagcag caacacccat gtcttcaccg 8640gctatttctg gagaacctga aagtccgtta caacctttta ctgagcaaat ggagaaggaa 8700gaatctaaag tgcctcaaaa taatcttttc agggatggca atgttaatga acatttaaga 8760gatattcgat attatagtgg aaaatctgtt cagctagatg gggatcaaaa gctaaaaggt 8820tcagactttg gagaagacta tccgtgtttt tcaaaaggag agaattttat gaaagcagct 8880aagttaaaaa gagatgcaga ttatactgaa acttatgaag tatctgctca agatgcagat 8940aataattttt ataaggtcat gttaatgaga cctgaaacat tatttggttt gtattatttt 9000gaaagtatca taaagaacat tatgagtgat cctagtaata ctgtttttct tagaaggttg 9060tgtgctttgg ctgttgaatg gaatggaagg ttaaaaggtt tcataccgga attgccagat 9120gataggcatg agtggttgag agatttaatt accctattag ctgccatttg taggtcatgt 9180gttacggttg atgaacaagt ggcagctata aacactagtc tagtagaaat ggcattaaat 9240ttttcttctg cagcttctgt aataccttct gcagctttag gtgtacaaac tagaagtatt 9300ttgagttcta tatgtaagga aattttacaa aatatgtgtc aaattggagt ttgtaaggat 9360aattatcgac cagctgtgca atattatgca gatcagcctg gaatctcaca tagtacatac 9420ttttctagtt tgagagacgt tctacaatca aacggaagaa atgtcttcta aggatgtggc 9480agagatctta tctggaaatg ctcctagatt gtcaaaggaa tttagaaata tgcccgtagc 9540taataaaatg attgagttgg agaaagcaat tgttcagcct aaaaagacag atactccaac 9600catgctttct ataattgtta aacaattagt tgatacaggg gctattttcc ctgaagaagc 9660ttctgctgtt tatagcaggt tgttggacag gcttgtaaag tttaactcta ttagaaatca 9720taataactta gaaggtcttg ttaatgatat acagcaaggg cagaaaagtg ttgtaatgtc 9780taatcttaaa gctaatagaa acatgtctaa tgttgttgta ttacagaatt tcttgcagca 9840gctgccaaaa actgtttcaa aaggccagca gaattatgac tcttttaaag gtttattgaa 9900acagtttgtg attgattata atcaatttat agaagtttat aaatcaggtc cggatacatt 9960tttacagtat aactttggtc cagctgtaca aaaaattaat ttaaatcaat cttttagaaa 10020tttgtcaaat ttatggggag ctgttgtgcg atctgaagat gatattccat ctttgtcagc 10080tttattagaa ccgcaaacga gatatttgtt gcttttactg tctcccatag ctatcgagca 10140gtattttaca agagatagct ttgtatggta tatgttgaaa ctatataaaa ataccgttgc 10200tcctccaatg agtactgagc cattagtaga gttgggtaat gttatagcta gtcttggacc 10260gagttatgat caattaaagt tgcagcaagg attgaattat ttggtaacta accaaagaca 10320agaatataaa ccatcagtac ctgacttgac taaagaagaa gaagcattac ttcgttattt 10380tcaaacttta cttagaacaa aagttgctgg tacaacacgt cagttaagac agtcagattt 10440agataatgtt attcaaaatg taaatcctgc tgctttccag ggcaatgtgg attttattaa 10500taggcttttt gattttttta gtaaaatctt gaaaataaat ccagattttt taactagaat 10560agtttatgat tctcaatgga aactacctcc agctttcttt ttgaagtctg taattactcc 10620tcaagatttg ttacaatttc ctcaacctaa aagaattcca gatcctaata tagttcaggt 10680tcctgtttct aatgttactg ttccagttcc agcgcccagg actaaattta aaatgccaca 10740acctgtgtcc aggccttcaa aaacagccta taggtctaaa tatcagtatc ctagtgaatc 10800tgatacagat actgactcag aaattgaggt atttggtaag ccttacggac caataaaacc 10860agctacaatc gacattgaca acttgtctgc tcaatttaaa agactgaaag gaaagggttt 10920agatatttct aattatatga gaagaaaagc aagaaatgtt aatgttagac catactgacc 10980aattcctggt tcattttaga tggaatcttc gaacactgcc actagaattt ttgctccaac 11040ggaagggaga aacagtataa tttacagcaa cttgcctcct gttcaagata caaccaaaat 11100attttatata gataacaagg ccattgatat agagtcatat aatcaagaga aagatcattc 11160taattattat actaatataa ttcaaacaca gaacatttca actattgatt caagtataca 11220gcaaattcag ttagatgaaa ggtctagatg gggaggagaa ctacatacaa gcttagtaac 11280atctgttatg aattgtacta aacattttaa ttcagataga tgtttagtga aaattcagac 11340tattaagagt ccacctacat ttgaatggaa agaattgaaa atacctgagg gaaactatgt 11400tttaaatgag tttattgatt tattaaatga aggtattact tctttatacc ttcagtatgg 11460caggcaacag ggtgtacttg aagaagacat aggaataaaa tttgatactc gcaattttga 11520aattggtaaa gatccaacta ctaatcttgt tactcctggt aaatacttgt ttaagggtta 11580tcatgctgat ataatacttc ttcctggttg ggctattgat ttttcttttt ctagattggg 11640taacatttta ggtattagaa aacgtgagac ttataaagct ggctttttga ttgaatatga 11700tgacttgaca aatggtaata ttccaccact gttggatgtt gctaactata agtctacaag 11760tcaagctaaa ccattattac aggatccatc tggcagatct taccacgtta tggatagtga 11820ttctaacaga cctgtgactg catataggtc ttttgttttg tcatataaca atgaaggtgc 11880tgcaaaatta aagtttttga tgtgtatgag tgatataacg gggggtctca atcagctgta 11940ttggtgtttg cctgattctt ataaaccgcc agtatctttt aagcaagaaa cgcaagtaga 12000taaactgcct gttgttggta tgcaactttt tccttttgtt tctaaatctg tgtattctgg 12060tgctgctgtt tacacacagt taattgaaca gcagactaat ttgacacaaa tttttaacag 12120atttcatgat aatgaaattt taaaacaagc tccatatgtg aatcaagttt tattggctga 12180aaatgtgccc ataaatgtta atcagggaac aataccaata ttttcaactc ttccaggagt 12240acagagagtg gttgtggaag acgataggag aagaactgta ccctacgtta ccaagtcact 12300tgctacagta tatccgaagg ttttgtctag caaaactttg caataatgca ttctgttgtt 12360tattctccag gggacagtag aggatggggt attggtaatt caagtatgcg agattattat 12420ttgataggtg gcgctttgca accgtctgat atttatactg ttagggttcg tgaacattgg 12480agacgtaaaa ggaggccaac tgctcaaact ggaaattctg ctgtaacccc acgacgtaga 12540agacggagaa caattgcaat tcaagtacca gctccaacta gagtactaag aaatagaata 12600gttacacctg ttgtgcctgc agttcctgta cctgctccta cagtttctgc tgtaccagta 12660cctgctgctc ctgtagctgt agctgctaag agacgtagag taggttagaa atgtttgaaa 12720atttagcacc cagaaaaggt ctaaaaaccg aaacacggaa tgtaaagttt agtaatgaat 12780tgagaggtgg ttttgttgtc tctgttttag ttcctttgct ttcttcttta ataggcgcag 12840ctcctgccat tgctggaact gtaattgcag ctagaaattc taagtagtgt tttttcttac 12900agatatgttt tcaaatttag ctccacgact tggacacaca tcattttcaa ctgtatctgt 12960tgggtctgct gaactgcgtg gaggaaagat taattggggc tctttaggtt cttccatttc 13020aaatgcttta agaacaactg gcagatattt aggccagaaa gctactaaat ttgcaaatag 13080taaaacattt agtgatatta aggccggtat tcaagatagt ggtttagtaa gaaatgtggc 13140aggattagca ggtcaaacat tgaattcttt agtcgatatt ggaaggttta aagttgaatc 13200tgaacttcaa aaattaagag atagagtatt aaatacaatt ccagcagatc agttagctca 13260aattttactg aactatcagc aaactcatga tcaggtgcct atgcctgtca caccaggtga 13320tgctattcct ttaccaccac cacctccagc tgctattgaa cctagaaaac gtccttatgt 13380tgaggaaata gacgataatc ctaacgatgc agaagtggtt attgacaccc ctgctttgtc 13440tactgttcct gctatacctg cacctcctcc tactgttgct tttgtacctt ctattaaacg 13500tcctagaatt aggggaactg gtgaatctga atggcaaact cacttgaata aaatgttggg 13560tcagggtgtt agatttacct caacaaatca atgttattaa ttttttagat ggacatatca 13620aatgctacgc caaaacttga tatattccac atagctggac cagatgcttc agaatatctt 13680tcagaaaatc tcgttaattt catctccagt acagaatcgt attttccaat taataaaaaa 13740tttagagaaa caattgtagc accaacaaaa ggtgtgacga cagaacaatc tcagaaattg 13800caagttaaaa ttgttccaac tttgacacaa gatttagaaa atagttttac tgctagattt 13860actattgctg ttggcgatgg tcgggttttg gatatgggaa gtacgtattt tgatattagg 13920ggtaacattg atcggggacc ttcatttaag ccatatggtg gtacagcata taatcctcta 13980gctccaaggt cagctcaatt taataatatt aaaactgtgg gtggtaaaac atatttgact 14040gctcaagcta ctaaattttt ttcaacatct ggaaatggtt gtgcagctgc taatactgaa 14100gcaagttcat ttacaaattt agttccttca cctaatactg gttcagcaga aagttctttt 14160gatcctacaa cagagggagc tagttgcaga gctataacac ttggcagttc tgtaacagat 14220gcaacttgtt atggagctta tacacctatt caaaatgcta atggttcaat tttacctcca 14280tctgttacgc ctgataaaaa atttgccgat gctggtaaat ctggcagtgt tacatgtact 14340gctgctattt gttgtgataa tgttactgta caatatccag atactagaat agttgcttat 14400gactctactg ataaaatagc aactagaatg ggtaacagaa ttaattatat tggatttaga 14460gataatttta taggtttgat gtattatgat aatggtgcac atagtggttc tttggctaca 14520gaaacaggag atataaattt ggtagaacaa ttgcaagata gaaatacaga aattagttat 14580caatatatgt tagcggattt gatgagtagg aatcattatt atagtcagtg gaatcaagct 14640gtagatgatt atgatttaaa tgttagagta cttacaaata ttggttatga agagggtcct 14700ccaggttact gttatccaag cacaggcatg ggcaactatc ctaatactgt catgtcggtt 14760gggacattag tggataataa tggtacaact gctacaacaa cgtcaaatac tgtagctgtg 14820atgggttttg gcagtgttcc tactatggaa attaacgttc aagcttattt gcaaaaatgt 14880tggatgtatg ctaacattgc agaatattta cctgataagt ataaaaaagc tattcaaggt 14940actagtgaaa ctgatccaac aacttatagt tatatgaata gtaggcttcc taatgtgaat 15000atggctgatc tctttacaca tattggcggg cgttatagtt tggatgtaat ggataatgtt 15060aatcctttta atcatcatag aaatagaggt ttgcaatata gaagtcaaat tttgggtaat 15120ggtagaaatg tccgttttca tattcaggta cctcagaaat tttttgctat taagaatcta 15180ttgttacttc ctggaactta tagttatgaa tggtggttca ggaaagatcc aaacttagta 15240ctacagtcta cgttgggaaa tgatttaaga aaagatggag caagcattca gtttagcagt 15300attagtcttt atgcgagttt ttttcctatg gatcacgcta cttgtagtga gcttatttta 15360atgcttagaa acgatcaaaa tgatcaaact tttatggatt atatgggtgc aaagaataat 15420ttgtatttag ttcctgctaa tcaaactaat gttcagattg aaataccttc tagagcttgg 15480acagcattta gaggctggag ttttaaccga attaaaactg ctgagacacc agctgtgtgg 15540tctacttatg atcttaattt taaatattct ggctcaatac cttatctaga tggtacattt 15600tatctttctc acacttttaa ctctatgtct attttgtttg attcagcaat aacatggcca 15660ggtaatgata gaatgttagt tccgaatttt tttgaaataa aaagagagat agatacggag 15720ggatacacta ctagtcagtc taatatgact aaagattggt atttgattca aatgtctgca 15780aattataacc aggggtatca cggttatagt tttccagcag ataaagtata cagacagtat 15840gattttatgt caaattttga ttctatgtct gttcaagtac cccggtcagg tctggcattt 15900ttgtttaatg aaaattataa cttgatagta aataattcag gatttttgcc cagtaggacg 15960gctccaattg ctggagttaa tgaaggccat ccttatccag caaactggcc agcgccatta 16020ataggtaata gtcctgatag tgttgttaca gttaggaaat ttttatgtga taagtattta 16080tggacaatac ctttttcaag caattttatg aatatgggtg aattgactga ccttggacag 16140agtttgctgt atactgagtc tgcacatagt ttgcaaataa catttaatgt tgatccaatg 16200cctgagccta cgtacattta cttactttat agtgtttttg attgtgttag ggtcaatcaa 16260cctaacaaaa attacttatc tgcagcttat ttcagaactc cttttgctac tggaactgct 16320tcagtataaa atggctggaa cttcaagttc agaattgatt acattagtac gttctttagg 16380tttgggttct tattttttag gagtttatga taaacacttt cctggttttt taaatgatcg 16440cagattagca tatgctattg taaatacggg tgattatatg tctggaggtt tacattggat 16500agcttttgct tacgacccta atggtcgaaa attttacatt tttgatccat ttggttggtc 16560aaaaaaggag ctttggaagt tttacaagtt tcagtatgat agaattgtta gaagaacagc 16620gttacagaat ggtagatgta ttaaattagt tagatctgta gatactgttc agtgtccttg 16680ttctgctgca tgtggtttgt attgtgtgtt atttttagct tcattttatt attttagaaa 16740ttctcctatg tataataatc ctattattga tgttgttact ggcgtgccgc atagtaagat 16800gaaatcttct tacggcatag ctatattaca ttgtaatcaa gaaagattgt ataactggct 16860gtattacaac tctgtatatt ttcgagataa tgaattggaa ataaagagaa atacaagaat 16920aaattctatt ttagttcatt atctctttat tgtattgttt ttatttgcgc gttaacaaaa 16980agcgtcatca tcactttcca cctcgtgttg agccacaata gcttgcttaa aagaatgctt 17040tttaggattg aatacaaaag caggcaacat tattttagta ggggagccat catctatggt 17100ttccttcagc ttagtatata tatctttgct aattttcata gcctgtctca catcaatcat 17160tgacaattta aaatcacaat gtttgtgagt ctgattatct ttatccttag catttctttt 17220aagtctaatg ggattgcaac attgaaacac aaaggtgtgt ttatacttgg cagttgcaag 17280aagcatatca tcatgacaac tctcagggtc tatgtcattc gctccaggta tttcaaaagc 17340agtcattttg cagatttgtc tgccaagctg aatgttttca tgaccatagt tacagaagca 17400ctttgtaact ataatcattt tttcagaaat ttcacatttc tttgctttag gaaacatagc 17460acttgtccaa tcaatattat gaagaaaggc agcttttgct ttttctttgt tgccaaaatt 17520cattccacaa gagtctggtg aatgaatagt tggccactga acattactat ctaatggaca 17580gactatattg ctataatttg ttagctttat cacgtctttt tgattttttt ctttctctaa 17640tctgccctca ccagctgtca gagctctcat tccttcctct gattgaggag acaggctgta 17700agttatgggt tttgaaagca taacagtgcc atgaaaacat ctaatgttat cttcatccca 17760attatgaacc cagatattgg ctccaaggca attataattg caaattaagc cgctatcatt 17820atacacaaaa ctgcttacga tacggccagc aaagtgataa aaagatttag cggaggaaat 17880cgtaagatta agatctggtt tattcttctt aacatatgtc tcaaccagtt tggcccagta 17940atcagaagta ggtaaaatat tcgcttcagc ttggcatgca aacaatgctt gtaatcctgt 18000agcgtattgc atagcttttt ggtgacagtt ttcaaattca tcattttcca cccgctgtct 18060cttaaggcaa ggctcctcct ctgagtctaa aaaaaattaa atttattaca tttaaatgca 18120aatagttttg catacgattc tgattggtct tacttaccaa ccaatgagcg ctggtcagaa 18180gacatgatct ataaaagagg aaaagagaga ggaaattcta aaattataat ggcttcgtct 18240gaggaggtcg tagactctgc agcgcaagaa ttcaatgaac ccttcccgcc agcaccagaa 18300acattaccag attcagaagt tgatatagaa cttatgaatc gtgacttggg tgagtttgaa 18360acaaattctt ttagcatcca cttaaggaga caagcacaat tgtgcaaatt ggctttacaa 18420gctaaattca aatatttacc agaatctgta gctgaaattg gagatgcatt cgaatcattc 18480atttttaatc caattactga atctgaccga aaacaacaag agcctagact caatttttac 18540cctccatttg ctgtgccaga acgaacagca acttacaata gcttttttca aattatgtct 18600ctaccattta gctgcttagc taacagatca ggtagtaaaa aatataagac tctaaaatca 18660attacaaaat ttgaagtctt acccaagttt gaatcagata tgtttgtgat ttcagactgt 18720cttgggtccg aagtctcagc aacagattct ctgccaagga aaacaaggtt ggttaattta 18780caatctgata acataagatt aatgtccatg aaagaaaaac tgaagcatgt aactcaattt 18840gcttatccag ccttgaacat tcctccaaaa atttataaaa ctctaattga gacactatat 18900aaacctattc aacagggaga ggatgatgaa tctgattatg tgttttcaga tgatgatgtt 18960agacaagtct ttatttcaaa tttagaggat tttgaaaaat ttactgatgg agagatagga 19020gaattaacaa attgtttcag aaaaaacttg cttcaggcaa tacagtatgt gctaccttta 19080aaacttatgc aaggtacttt tagacatccg tgctttgtaa agaaattaca agagatgtta 19140cattatactt ttcatcatgg ctatatcaag ttaattagtt ctattacggg ccacaatttg 19200agtaaatata taacttttca ctgcatgaca tatgagaata acaataacaa tccaaatctt 19260cacacaacat tagatttgaa tgatggtgaa gattatatgg ttgatacaat ttttttatac 19320ttgataatga cttggcagac tgcaatgggt gtgtggcaac aaaatatcaa tgagaagaat 19380ttagctagta tgaaagattt tttaactaaa aacggaccaa aattgatttt gtgtcgtgat 19440tcagatagca tggctgatat gctagcagat tggataacag atggcggagt cttgcttcag 19500atttttaggg atgctttacc agattttatg tcacagactc aattgaataa ctttagaaca 19560tttattttag cgagaagtaa tatagtgagc tgtatggttt caacagtagt taaagatttt 19620gtaccattag attttaaaga atctccacca caattgtggc cacatgttta ctgcttgaga 19680ctgtcttatt ttttctacaa tcatggagat tatcaacaaa ttttttattg ggacgataat 19740aaacctacag aaaatgaaat tttttgttat tgcaatcttt gtgctcctca tagaacacca 19800atgctgaaca cagctttaca caatgaaatt ttagcaattg ggtcgtttga cttttttgtt 19860ccaagtagtg atggtaaagg tggagaaaga gttacattaa ctccgggatt atgggctaat 19920aaatttttga atcattttgt aagttctgaa tattttccat ttgaagttaa aaaatatgta 19980gaccatccag aatgtttcaa aatacctcct acagcatgtg taattactaa gcctgagatt 20040ttaagtagtt tgaaagagat aaagaagagg agagaaaagt ttttaattga aaaaggttct 20100ggtatttatt tggatcctca aacgggagat aacttaagtg atgctaaaat tgtttcacag 20160cccagaagag gcagcagtgg cggaaaaaca gaaaaagaag aaacggcaaa gaagaatccc 20220aggtagttat tctaaatgga agcaatactg cacagatgta aaagaagcaa ttgcagttgt 20280cggaagacag ccagtttgtg ttaagagata tctcacagca aagggcatac aaattccttc 20340atcaaccatt aattattatg taaacaagtt tataagttgt gaagaagata gtaaattttc 20400ctttttttaa tttataggcg agcccataca tgaaggaaca tatcaagaaa tattaaaact 20460tagaaaagaa atttggatta ctgtgagaga cttaaaagat tatctatata ataatgagat 20520aaacgaagaa attcatatac agaatagaac tttaaattct atcttagcta agcattctac 20580atgttcagat ttaaatgcat tatttaagat gcatttggat gctaaggctt tacaatatca 20640atatgagaaa aagctccgca ccatacctga taagaagtct cagaagaaaa agagatagct 20700tctaccctga cagcaaattt tatagagaag acctccccta gtcacctgac caagcttggc 20760gggcaatatg gatcccgttc cgttagaata tatttggcag tacaaccctg tgactggtag 20820agttggagga gcaaatcaga attacggaca gaggattaat gttttgcata caaatcgtta 20880cctctacaac agaatgcaga atgtgcagaa aaaaagcaac gaaagagcca ctgaaagagg 20940attactatct ctgaagggag gaagcacatt gccgactatt gcagaagatg aacctgccca 21000gttgaactcg gctatagttc gcatggcagg attgaatgac ttgaacacgg ttcaagcacc 21060ggattcatct aatctgcaaa acctggccaa cattgcactt gaagctgctg aacacaacaa 21120agctacctca tctctcctaa caacgaagaa gtttgttgaa gagtttcctc ctgttgttta 21180cgaaaatcct ttttctggct ctaattttgc ttatgaattt aatcctctgt attcaccgtc 21240cgggaatgag ttctccaatc ctccatttaa attcaccgga ggtgctatta gcctcacagg 21300acaacaccca gtcctctcgg gaggtgctgt tatcttatca ggacagaatc cggtactgga 21360caaagcataa acatgagtgt caatctatta attgtatgac ggatgttgtt aatgtaatgg 21420atcttgtttt tgattgtgat tatcctgagt ttaaatctgg aagcagtgtt atgttttctc 21480atcgcttttc ttacttaaca gattgcaaaa ataaaacaat attaccgctt gttgatgaaa 21540attgtgaatt aatgctagtt aatgatgaga tatgtgtagt tgtaaaatgt tactgtgatg 21600aagaattttc agatcattgt cttactatta acttgaaatt aatgtgtacg ttagtaatga 21660aattaataga tggggcttac ccattgccgc cttcgcctgc agatttacct gcttcagctt 21720gtgttgcaac agcgctaaaa aatgagaaca agagagactt tttacataat agcttttgca 21780ataaatgcag aataccagta ttgctttgct tatgtactga aaatgcttta acgaaggaag 21840aatttatgca tcaattagtt aattttccat tatgtagtaa ctgttttaaa ggtaatagtg 21900attgtagttg tgcattttat atctgtaagt tgtattcttt ttatcgaagc agagtttcct 21960ccggtttagt taggaagaag ttaaatgatg ttaagaggtt agcatattta ggatcatgta 22020acgttgatat gtgtattaac tgtggaagaa gtcttaagga ttgtttgtgt tctgaagctt 22080atcagttatt atataagaaa tgcctttctt attgatagat tgtttacgaa gcagaagcag 22140agtttctcct ccaatttagt aagattatta ttactgtgcc tagatattac tatgtgttca 22200ggaagaagtt ttacggattg tctgtgctct tacaagaaat aaaaaatttg acatttactt 22260acctgtctac ctggtcatgt attgatggga agagagtttc caaaattaag tctgagtttt 22320ggatacagta tcgaattaac caatagagac acgtttgaca cagaagctgt gttgctgacg 22380gtctatattc tgtttgtatt cctttttcca ttccatccac agaatcttca gatacagttt 22440ggatgttttg tttaagtaaa ttctgattga tttctcttgg tttgcaacag gaacaacatt 22500tgctgcattt attgcccatg gctactcctg gaaagcgttc tgcagaggaa ccagatcaac 22560agaccttgaa aaagtcaaaa caatctgacc aaagtcaggg tttaaatcta gcatatcctt 22620ttgataaaat aacagaattt gaagcaacac ctccctttat tcatgttggg caaggcttag 22680acatatcaga tttatcgtta aatatgagaa ttggcaaagg attaaagttt gaaaatggta 22740atctagttgt atcagatcaa cagtataatg ttacaccacc tttaattgca gatcagtcaa 22800cattaggttt aaagtataat ccggatgttc tttctttaac acattcaggt gctttaactt 22860tgccaactat tcaacatccc ctccaggctt cagctggaaa atttgaactt gctttgtcat 22920caggtttaaa atctgatgat caaggtttaa ctttagattt ggatcctgta ttttctacag 22980aatcatcaaa atttttgctt aattgttcat tgccgttaga taagaatagt gacaagttaa 23040cgttaaaatt tggtaatggt cttggattga ataatgacca gctagagaat actatgactt 23100ataatcttcc tttaaaacgt gatggaacta atgttagtct ttcatttgga actaatttca 23160aaatattgaa tgagatgtta gatttaaatc ttgtggcacc tatgtctaat tcagcaggag 23220gattagcatt gcaatttaaa agccctttgt cagcagatga tggtatttta tcaattaaaa 23280cagatacatc tttgggtata acaggaaata aattaggaat aagattggcc cctaacagtg 23340gtctgcaaat aacaccaaat ggtctagcag ttagtgttaa tgctgtgcaa attctaagta 23400gtcctttaat tactgcagcg tctataggcc caccaacaac aatggttact ggaacagtgt 23460caccgggcag agcaacaaat ggtcaatttg taaccaaaac tgctaaagtt ttacgttata 23520aatttgtgag atgggatgct ctgttaatca tacagtttat agataacata ggtgtaatag 23580aaaaccctac cttttatcgt aacaaaagta ttgaattaag atctgctgat ttcttgagtc 23640ctacgttaaa taatacatat atagtgccat tgaatggagg ggtaagggta gaatcaccta 23700ctattcctgt acaattagaa gttatacttg aaaacaattc ctctttcatt caagtagggt 23760ttgttaggtt aacagttaag aatggtaacc ctcatatgat tattcagtgt aatcctgtac 23820ctgggaatat taaaatgata aagataaaat ctgtaatgct ttttacttgt ttgataggct 23880gatgaaataa actagtgatg caactttcgg ttttagtgac tcactttcga tttaaacacc 23940tgcaggaagt ttttgctttc ttttttatcc tacatacggt tagaacgtta gattctcaca 24000tatttgactt tgcaggtgcg cagagcggga aatttttatt ttgcatattt tttattttta 24060tgtctggctt taaccacaac tcctttacga ttggctatta aaatgtcaag ttaagacaag 24120tgtgaatttt ctcattagta atgctgtggc gcgaaacaca tattttttta tctaaagcct 24180tcgagtaacc acaactttta tatgattggc tacttaagtt tcagttaggg taaatgtgaa 24240gtttctcatg agaaatgcta tgccgccaaa aaaaaattta catcttgctg cagttcaaag 24300ggtaaattct ttttccacta acatttcatg tctttcaagc gggaaaatgc ttggtcgagc 24360caaagaactt aaatagttag tttgtggcca tggtttgaat gcttcactga ttaaatagac 24420agattgctat tgagttagag cgggtaaaac tgagccaaga atctgagcgt gtagtataaa 24480aaggactcac acttacacac ttacaatcat gcctctgact tggtggttgc aagctgacat 24540tcacttcaat gaagatgacc aatttcagca gaacttaagt cttactcttc aagcaatggc 24600tgagaacaaa gaagaaaaag actgtaaatt caacgtaact attcataagg aaattgaacc 24660tgaattgaat actatttttg atactcaaat ggatacctgg ttatcttgcg gctttactaa 24720aatgactgtt ttttcaactg gtaaaggaga tatatttttg cgcattttat ttagaacttg 24780tgctatacct tttttactgc tggagtggaa ggaagaaaat taaaatctaa aattgaacaa 24840tcaatgtacg agacctttgg aagctgccta tctatggact ttattaaggt tgcttctgtt 24900gctgtgcaag gctacgatct gcctgctatc aactgtaatg ttggaaaacc tctcgtgttg 24960ctggtggcca cgcaagatga accgaatttt cgtgaaatac ctggaccata tatcaaagat 25020gtgtggatgc gtcactgtta cacaccaccg gaattaacgc ctgatgaaga tgatgacctt 25080tgtacaccat tagccagtca tttctcatat gattttgatc catcaattgt gcctgatatt 25140gccttgttag acctttttga ataaaaaata cttaaacaga aaatcgtgta atgtatttat 25200tatgcgaaaa cagtaccata aggtaatgag tggtcttctc ctaccaaacg cacgtggtca 25260aagaaattaa ctccattact tatacactgn aaaagcacac cacgcaagta cacaaggcca 25320gtagtcaatg ttttccaagt catacagtgc aatccgacgc tctgtataaa cattaaccgt 25380gttactcgaa atcaacatta ggccacttac catcaagcat gtagtcgaaa catgtaatgg 25440aaatccgaga ctcatttcta gtgttacaac atcaaagtca tcggaacagt ttaccaaaaa 25500ttatattcag aaaagtttaa atgtacccgt attttccaac atgatggatt catcgtcttc 25560ggtctttaaa acatacacga accggagttg atgatggtgt aggcatcgtt tctcgcgagc 25620ctgatgagaa cgggttcgta gaatgtactt ttggcacttg tattcttctg tcttctactt 25680cattatcaga ttctccaaca tccatctaca acaaaaatgg agggtaaatt tttgccaact 25740aatgcacaat aagctatgaa catcattatg acagatgcag taaatatgac ggtatattag 25800tgagcggtta aaaagtatta tcattgcagc gtgtctgcct gctcttatct gtagattgtg 25860ttttagaccg ggaggcattt tttccagtat taacagcgaa tgaatgtagt aaactacagg 25920ttccggcatc ggaatcacga aaggcatcgg catttcaatt tcggcattca tgtcctataa 25980aatacgattt aaaacatata tagtctctat acaacgttta aaggattgat ttgtgaaaaa 26040aaagattata aagacttacg atttgagagg attgagaaga atttgaaaga actgccgcgg 26100atcctgcttg tgattcttta aaatggcgat tatccgcctt cttttccggt tctctaacga 26160acacgtgtcc taaagaggat gtcatcatat ttgaccatgt aatatcctgt tcaattttgt 26220catctgaaag tcgaaatccc ctcccaagca tgcggtatat atattgattg 26270 2 900 DNA hemorrhagic enteritis virus 2atgaatacca ttatgaaagc aatgcaaaca gatagaggta gactagaagc agcaacaccc 60atgtcttcac cggctatttc tggagaacct gaaagtccgt tacaaccttt tactgagcaa 120atggagaagg aagaatctaa agtgcctcaa aataatcttt tcagggatgg caatgttaat 180gaacatttaa gagatattcg atattatagt ggaaaatctg ttcagctaga tggggatcaa 240aagctaaaag gttcagactt tggagaagac tatccgtgtt tttcaaaagg agagaatttt 300atgaaagcag ctaagttaaa aagagatgca gattatactg aaacttatga agtatctgct 360caagatgcag ataataattt ttataaggtc atgttaatga gacctgaaac attatttggt 420ttgtattatt ttgaaagtat cataaagaac attatgagtg atcctagtaa tactgttttt 480cttagaaggt tgtgtgcttt ggctgttgaa tggaatggaa ggttaaaagg tttcataccg 540gaattgccag atgataggca tgagtggttg agagatttaa ttaccctatt agctgccatt 600tgtaggtcat gtgttacggt tgatgaacaa gtggcagcta taaacactag tctagtagaa 660atggcattaa atttttcttc tgcagcttct gtaatacctt ctgcagcttt aggtgtacaa 720actagaagta ttttgagttc tatatgtaag gaaattttac aaaatatgtg tcaaattgga 780gtttgtaagg ataattatcg accagctgtg caatattatg cagatcagcc tggaatctca 840catagtacat acttttctag tttgagagac gttctacaat caaacggaag aaatgtcttc 900 3 300 PRT hemorrhagic enteritis virus 3Met Asn Thr Ile Met Lys Ala Met Gln Thr Asp Arg Gly Arg Leu Glu1 5 10 15Ala Ala Thr Pro Met Ser Ser Pro Ala Ile Ser Gly Glu Pro Glu Ser 20 25 30Pro Leu Gln Pro Phe Thr Glu Gln Met Glu Lys Glu Glu Ser Lys Val 35 40 45Pro Gln Asn Asn Leu Phe Arg Asp Gly Asn Val Asn Glu His Leu Arg 50 55 60Asp Ile Arg Tyr Tyr Ser Gly Lys Ser Val Gln Leu Asp Gly Asp Gln65 70 75 80Lys Leu Lys Gly Ser Asp Phe Gly Glu Asp Tyr Pro Cys Phe Ser Lys 85 90 95Gly Glu Asn Phe Met Lys Ala Ala Lys Leu Lys Arg Asp Ala Asp Tyr 100 105 110Thr Glu Thr Tyr Glu Val Ser Ala Gln Asp Ala Asp Asn Asn Phe Tyr 115 120 125Lys Val Met Leu Met Arg Pro Glu Thr Leu Phe Gly Leu Tyr Tyr Phe 130 135 140Glu Ser Ile Ile Lys Asn Ile Met Ser Asp Pro Ser Asn Thr Val Phe145 150 155 160Leu Arg Arg Leu Cys Ala Leu Ala Val Glu Trp Asn Gly Arg Leu Lys 165 170 175Gly Phe Ile Pro Glu Leu Pro Asp Asp Arg His Glu Trp Leu Arg Asp 180 185 190Leu Ile Thr Leu Leu Ala Ala Ile Cys Arg Ser Cys Val Thr Val Asp 195 200 205Glu Gln Val Ala Ala Ile Asn Thr Ser Leu Val Glu Met Ala Leu Asn 210 215 220Phe Ser Ser Ala Ala Ser Val Ile Pro Ser Ala Ala Leu Gly Val Gln225 230 235 240Thr Arg Ser Ile Leu Ser Ser Ile Cys Lys Glu Ile Leu Gln Asn Met 245 250 255Cys Gln Ile Gly Val Cys Lys Asp Asn Tyr Arg Pro Ala Val Gln Tyr 260 265 270Tyr Ala Asp Gln Pro Gly Ile Ser His Ser Thr Tyr Phe Ser Ser Leu 275 280 285Arg Asp Val Leu Gln Ser Asn Gly Arg Asn Val Phe 290 295 300 4 1515 DNA hemorrhagic enteritis virus 4atgtcttcta aggatgtggc agagatctta tctggaaatg ctcctagatt gtcaaaggaa 60tttagaaata tgcccgtagc taataaaatg attgagttgg agaaagcaat tgttcagcct 120aaaaagacag atactccaac catgctttct ataattgtta aacaattagt tgatacaggg 180gctattttcc ctgaagaagc ttctgctgtt tatagcaggt tgttggacag gcttgtaaag 240tttaactcta ttagaaatca taataactta gaaggtcttg ttaatgatat acagcaaggg 300cagaaaagtg ttgtaatgtc taatcttaaa gctaatagaa acatgtctaa tgttgttgta 360ttacagaatt tcttgcagca gctgccaaaa actgtttcaa aaggccagca gaattatgac 420tcttttaaag gtttattgaa acagtttgtg attgattata atcaatttat agaagtttat 480aaatcaggtc cggatacatt tttacagtat aactttggtc cagctgtaca aaaaattaat 540ttaaatcaat cttttagaaa tttgtcaaat ttatggggag ctgttgtgcg atctgaagat 600gatattccat ctttgtcagc tttattagaa ccgcaaacga gatatttgtt gcttttactg 660tctcccatag ctatcgagca gtattttaca agagatagct ttgtatggta tatgttgaaa 720ctatataaaa ataccgttgc tcctccaatg agtactgagc cattagtaga gttgggtaat 780gttatagcta gtcttggacc gagttatgat caattaaagt tgcagcaagg attgaattat 840ttggtaacta accaaagaca agaatataaa ccatcagtac ctgacttgac taaagaagaa 900gaagcattac ttcgttattt tcaaacttta cttagaacaa aagttgctgg tacaacacgt 960cagttaagac agtcagattt agataatgtt attcaaaatg taaatcctgc tgctttccag 1020ggcaatgtgg attttattaa taggcttttt gattttttta gtaaaatctt gaaaataaat 1080ccagattttt taactagaat agtttatgat tctcaatgga aactacctcc agctttcttt 1140ttgaagtctg taattactcc tcaagatttg ttacaatttc ctcaacctaa aagaattcca 1200gatcctaata tagttcaggt tcctgtttct aatgttactg ttccagttcc agcgcccagg 1260actaaattta aaatgccaca acctgtgtcc aggccttcaa aaacagccta taggtctaaa 1320tatcagtatc ctagtgaatc tgatacagat actgactcag aaattgaggt atttggtaag 1380ccttacggac caataaaacc agctacaatc gacattgaca acttgtctgc tcaatttaaa 1440agactgaaag gaaagggttt agatatttct aattatatga gaagaaaagc aagaaatgtt 1500aatgttagac catac 1515 5 505 PRT hemorrhagic enteritis virus 5Met Ser Ser Lys Asp Val Ala Glu Ile Leu Ser Gly Asn Ala Pro Arg1 5 10 15Leu Ser Lys Glu Phe Arg Asn Met Pro Val Ala Asn Lys Met Ile Glu 20 25 30Leu Glu Lys Ala Ile Val Gln Pro Lys Lys Thr Asp Thr Pro Thr Met 35 40 45Leu Ser Ile Ile Val Lys Gln Leu Val Asp Thr Gly Ala Ile Phe Pro 50 55 60Glu Glu Ala Ser Ala Val Tyr Ser Arg Leu Leu Asp Arg Leu Val Lys65 70 75 80Phe Asn Ser Ile Arg Asn His Asn Asn Leu Glu Gly Leu Val Asn Asp 85 90 95Ile Gln Gln Gly Gln Lys Ser Val Val Met Ser Asn Leu Lys Ala Asn 100 105 110Arg Asn Met Ser Asn Val Val Val Leu Gln Asn Phe Leu Gln Gln Leu 115 120 125Pro Lys Thr Val Ser Lys Gly Gln Gln Asn Tyr Asp Ser Phe Lys Gly 130 135 140Leu Leu Lys Gln Phe Val Ile Asp Tyr Asn Gln Phe Ile Glu Val Tyr145 150 155 160Lys Ser Gly Pro Asp Thr Phe Leu Gln Tyr Asn Phe Gly Pro Ala Val 165 170 175Gln Lys Ile Asn Leu Asn Gln Ser Phe Arg Asn Leu Ser Asn Leu Trp 180 185 190Gly Ala Val Val Arg Ser Glu Asp Asp Ile Pro Ser Leu Ser Ala Leu 195 200 205Leu Glu Pro Gln Thr Arg Tyr Leu Leu Leu Leu Leu Ser Pro Ile Ala 210 215 220Ile Glu Gln Tyr Phe Thr Arg Asp Ser Phe Val Trp Tyr Met Leu Lys225 230 235 240Leu Tyr Lys Asn Thr Val Ala Pro Pro Met Ser Thr Glu Pro Leu Val 245 250 255Glu Leu Gly Asn Val Ile Ala Ser Leu Gly Pro Ser Tyr Asp Gln Leu 260 265 270Lys Leu Gln Gln Gly Leu Asn Tyr Leu Val Thr Asn Gln Arg Gln Glu 275 280 285Tyr Lys Pro Ser Val Pro Asp Leu Thr Lys Glu Glu Glu Ala Leu Leu 290 295 300Arg Tyr Phe Gln Thr Leu Leu Arg Thr Lys Val Ala Gly Thr Thr Arg305 310 315 320Gln Leu Arg Gln Ser Asp Leu Asp Asn Val Ile Gln Asn Val Asn Pro 325 330 335Ala Ala Phe Gln Gly Asn Val Asp Phe Ile Asn Arg Leu Phe Asp Phe 340 345 350Phe Ser Lys Ile Leu Lys Ile Asn Pro Asp Phe Leu Thr Arg Ile Val 355 360 365Tyr Asp Ser Gln Trp Lys Leu Pro Pro Ala Phe Phe Leu Lys Ser Val 370 375 380Ile Thr Pro Gln Asp Leu Leu Gln Phe Pro Gln Pro Lys Arg Ile Pro385 390 395 400Asp Pro Asn Ile Val Gln Val Pro Val Ser Asn Val Thr Val Pro Val 405 410 415Pro Ala Pro Arg Thr Lys Phe Lys Met Pro Gln Pro Val Ser Arg Pro 420 425 430Ser Lys Thr Ala Tyr Arg Ser Lys Tyr Gln Tyr Pro Ser Glu Ser Asp 435 440 445Thr Asp Thr Asp Ser Glu Ile Glu Val Phe Gly Lys Pro Tyr Gly Pro 450 455 460Ile Lys Pro Ala Thr Ile Asp Ile Asp Asn Leu Ser Ala Gln Phe Lys465 470 475 480Arg Leu Lys Gly Lys Gly Leu Asp Ile Ser Asn Tyr Met Arg Arg Lys 485 490 495Ala Arg Asn Val Asn Val Arg Pro Tyr 500 505 6 1344 DNA hemorrhagic enteritis virus 6atggaatctt cgaacactgc cactagaatt tttgctccaa cggaagggag aaacagtata 60atttacagca acttgcctcc tgttcaagat acaaccaaaa tattttatat agataacaag 120gccattgata tagagtcata taatcaagag aaagatcatt ctaattatta tactaatata 180attcaaacac agaacatttc aactattgat tcaagtatac agcaaattca gttagatgaa 240aggtctagat ggggaggaga actacataca agcttagtaa catctgttat gaattgtact 300aaacatttta attcagatag atgtttagtg aaaattcaga ctattaagag tccacctaca 360tttgaatgga aagaattgaa aatacctgag ggaaactatg ttttaaatga gtttattgat 420ttattaaatg aaggtattac ttctttatac cttcagtatg gcaggcaaca gggtgtactt 480gaagaagaca taggaataaa atttgatact cgcaattttg aaattggtaa agatccaact 540actaatcttg ttactcctgg taaatacttg tttaagggtt atcatgctga tataatactt 600cttcctggtt gggctattga tttttctttt tctagattgg gtaacatttt aggtattaga 660aaacgtgaga cttataaagc tggctttttg attgaatatg atgacttgac aaatggtaat 720attccaccac tgttggatgt tgctaactat aagtctacaa gtcaagctaa accattatta 780caggatccat ctggcagatc ttaccacgtt atggatagtg attctaacag acctgtgact 840gcatataggt cttttgtttt gtcatataac aatgaaggtg ctgcaaaatt aaagtttttg 900atgtgtatga gtgatataac ggggggtctc aatcagctgt attggtgttt gcctgattct 960tataaaccgc cagtatcttt taagcaagaa acgcaagtag ataaactgcc tgttgttggt 1020atgcaacttt ttccttttgt ctttaaatct gtgtattctg gtgctgctgt ttacacacag 1080ttaattgaac agcagactaa tttgacacaa atttttaaca gatttcatga taatgaaatt 1140ttaaaacaag ctccatatgt gaatcaagtt ttattggctg aaaatgtgcc cataaatgtt 1200aatcagggaa caataccaat attttcaact cttccaggag tacagagagt ggttgtggaa 1260gacgatagga gaagaactgt accctacgtt accaagtcac ttgctacagt atatccgaag 1320gttttgtcta gcaaaacttt gcaa 1344 7 448 PRT hemorrhagic enteritis virus 7Met Glu Ser Ser Asn Thr Ala Thr Arg Ile Phe Ala Pro Thr Glu Gly1 5 10 15Arg Asn Ser Ile Ile Tyr Ser Asn Leu Pro Pro Val Gln Asp Thr Thr 20 25 30Lys Ile Phe Tyr Ile Asp Asn Lys Ala Ile Asp Ile Glu Ser Tyr Asn 35 40 45Gln Glu Lys Asp His Ser Asn Tyr Tyr Thr Asn Ile Ile Gln Thr Gln 50 55 60Asn Ile Ser Thr Ile Asp Ser Ser Ile Gln Gln Ile Gln Leu Asp Glu65 70 75 80Arg Ser Arg Trp Gly Gly Glu Leu His Thr Ser Leu Val Thr Ser Val 85 90 95Met Asn Cys Thr Lys His Phe Asn Ser Asp Arg Cys Leu Val Lys Ile 100 105 110Gln Thr Ile Lys Ser Pro Pro Thr Phe Glu Trp Lys Glu Leu Lys Ile 115 120 125Pro Glu Gly Asn Tyr Val Leu Asn Glu Phe Ile Asp Leu Leu Asn Glu 130 135 140Gly Ile Thr Ser Leu Tyr Leu Gln Tyr Gly Arg Gln Gln Gly Val Leu145 150 155 160Glu Glu Asp Ile Gly Ile Lys Phe Asp Thr Arg Asn Phe Glu Ile Gly 165 170 175Lys Asp Pro Thr Thr Asn Leu Val Thr Pro Gly Lys Tyr Leu Phe Lys 180 185 190Gly Tyr His Ala Asp Ile Ile Leu Leu Pro Gly Trp Ala Ile Asp Phe 195 200 205Ser Phe Ser Arg Leu Gly Asn Ile Leu Gly Ile Arg Lys Arg Glu Thr 210 215 220Tyr Lys Ala Gly Phe Leu Ile Glu Tyr Asp Asp Leu Thr Asn Gly Asn225 230 235 240Ile Pro Pro Leu Leu Asp Val Ala Asn Tyr Lys Ser Thr Ser Gln Ala 245 250 255Lys Pro Leu Leu Gln Asp Pro Ser Gly Arg Ser Tyr His Val Met Asp 260 265 270Ser Asp Ser Asn Arg Pro Val Thr Ala Tyr Arg Ser Phe Val Leu Ser 275 280 285Tyr Asn Asn Glu Gly Ala Ala Lys Leu Lys Phe Leu Met Cys Met Ser 290 295 300Asp Ile Thr Gly Gly Leu Asn Gln Leu Tyr Trp Cys Leu Pro Asp Ser305 310 315 320Tyr Lys Pro Pro Val Ser Phe Lys Gln Glu Thr Gln Val Asp Lys Leu 325 330 335Pro Val Val Gly Met Gln Leu Phe Pro Phe Val Phe Lys Ser Val Tyr 340 345 350Ser Gly Ala Ala Val Tyr Thr Gln Leu Ile Glu Gln Gln Thr Asn Leu 355 360 365Thr Gln Ile Phe Asn Arg Phe His Asp Asn Glu Ile Leu Lys Gln Ala 370 375 380Pro Tyr Val Asn Gln Val Leu Leu Ala Glu Asn Val Pro Ile Asn Val385 390 395 400Asn Gln Gly Thr Ile Pro Ile Phe Ser Thr Leu Pro Gly Val Gln Arg 405 410 415Val Val Val Glu Asp Asp Arg Arg Arg Thr Val Pro Tyr Val Thr Lys 420 425 430Ser Leu Ala Thr Val Tyr Pro Lys Val Leu Ser Ser Lys Thr Leu Gln 435 440 445 8 360 DNA hemorrhagic enteritis virus 8atgcattctg ttgtttattc tccaggggac agtagaggat ggggtattgg taattcaagt 60atgcgagatt attatttgat aggtggcgct ttgcaaccgt ctgatattta tactgttagg 120gttcgtgaac attggagacg taaaaggagg ccaactgctc aaactggaaa ttctgctgta 180accccacgac gtagaagacg gagaacaatt gcaattcaag taccagctcc aactagagta 240ctaagaaata gaatagttac acctgttgtg cctgcagttc ctgtacctgc tcctacagtt 300tctgctgtac cagtacctgc tgctcctgta gctgtagctg ctaagagacg tagagtaggt 360 9 120 PRT hemorrhagic enteritis virus 9Met His Ser Val Val Tyr Ser Pro Gly Asp Ser Arg Gly Trp Gly Ile1 5 10 15Gly Asn Ser Ser Met Arg Asp Tyr Tyr Leu Ile Gly Gly Ala Leu Gln 20 25 30Pro Ser Asp Ile Tyr Thr Val Arg Val Arg Glu His Trp Arg Arg Lys 35 40 45Arg Arg Pro Thr Ala Gln Thr Gly Asn Ser Ala Val Thr Pro Arg Arg 50 55 60Arg Arg Arg Arg Thr Ile Ala Ile Gln Val Pro Ala Pro Thr Arg Val65 70 75 80Leu Arg Asn Arg Ile Val Thr Pro Val Val Pro Ala Val Pro Val Pro 85 90 95Ala Pro Thr Val Ser Ala Val Pro Val Pro Ala Ala Pro Val Ala Val 100 105 110Ala Ala Lys Arg Arg Arg Val Gly 115 120 10 174 DNA hemorrhagic enteritis virus 10atgtttgaaa atttagcacc cagaaaaggt ctaaaaaccg aaacacggaa tgtaaagttt 60agtaatgaat tgagaggtgg ttttgttgtc tctgttttag ttcctttgct ttcttcttta 120ataggcgcag ctcctgccat tgctggaact gtaattgcag ctagaaattc taag 174 11 58 PRT hemorrhagic enteritis virus 11Met Phe Glu Asn Leu Ala Pro Arg Lys Gly Leu Lys Thr Glu Thr Arg1 5 10 15Asn Val Lys Phe Ser Asn Glu Leu Arg Gly Gly Phe Val Val Ser Val 20 25 30Leu Val Pro Leu Leu Ser Ser Leu Ile Gly Ala Ala Pro Ala Ile Ala 35 40 45Gly Thr Val Ile Ala Ala Arg Asn Ser Lys 50 55 12 693 DNA hemorrhagic enteritis virus 12atgttttcaa atttagctcc acgacttgga cacacatcat tttcaactgt atctgttggg 60tctgctgaac tgcgtggagg aaagattaat tggggctctt taggttcttc catttcaaat 120gctttaagaa caactggcag atatttaggc cagaaagcta ctaaatttgc aaatagtaaa 180acatttagtg atattaaggc cggtattcaa gatagtggtt tagtaagaaa tgtggcagga 240ttagcaggtc aaacattgaa ttctttagtc gatattggaa ggtttaaagt tgaatctgaa 300cttcaaaaat taagagatag agtattaaat acaattccag cagatcagtt agctcaaatt 360ttactgaact atcagcaaac tcatgatcag gtgcctatgc ctgtcacacc aggtgatgct 420attcctttac caccaccacc tccagctgct attgaaccta gaaaacgtcc ttatgttgag 480gaaatagacg ataatcctaa cgatgcagaa gtggttattg acacccctgc tttgtctact 540gttcctgcta tacctgcacc tcctcctact gttgcttttg taccttctat taaacgtcct 600agaattaggg gaactggtga atctgaatgg caaactcact tgaataaaat gttgggtcag 660ggtgttagat ttacctcaac aaatcaatgt tat 693 13 231 PRT hemorrhagic enteritis virus 13Met Phe Ser Asn Leu Ala Pro Arg Leu Gly His Thr Ser Phe Ser Thr1 5 10 15Val Ser Val Gly Ser Ala Glu Leu Arg Gly Gly Lys Ile Asn Trp Gly 20 25 30Ser Leu Gly Ser Ser Ile Ser Asn Ala Leu Arg Thr Thr Gly Arg Tyr 35 40 45Leu Gly Gln Lys Ala Thr Lys Phe Ala Asn Ser Lys Thr Phe Ser Asp 50 55 60Ile Lys Ala Gly Ile Gln Asp Ser Gly Leu Val Arg Asn Val Ala Gly65 70 75 80Leu Ala Gly Gln Thr Leu Asn Ser Leu Val Asp Ile Gly Arg Phe Lys 85 90 95Val Glu Ser Glu Leu Gln Lys Leu Arg Asp Arg Val Leu Asn Thr Ile 100 105 110Pro Ala Asp Gln Leu Ala Gln Ile Leu Leu Asn Tyr Gln Gln Thr His 115 120 125Asp Gln Val Pro Met Pro Val Thr Pro Gly Asp Ala Ile Pro Leu Pro 130 135 140Pro Pro Pro Pro Ala Ala Ile Glu Pro Arg Lys Arg Pro Tyr Val Glu145 150 155 160Glu Ile Asp Asp Asn Pro Asn Asp Ala Glu Val Val Ile Asp Thr Pro 165 170 175Ala Leu Ser Thr Val Pro Ala Ile Pro Ala Pro Pro Pro Thr Val Ala 180 185 190Phe Val Pro Ser Ile Lys Arg Pro Arg Ile Arg Gly Thr Gly Glu Ser 195 200 205Glu Trp Gln Thr His Leu Asn Lys Met Leu Gly Gln Gly Val Arg Phe 210 215 220Thr Ser Thr Asn Gln Cys Tyr225 230 14 2718 DNA hemorrhagic enteritis virus 14atggacatat caaatgctac gccaaaactt gatatattcc acatagctgg accagatgct 60tcagaatatc tttcagaaaa tctcgttaat ttcatctcca gtacagaatc gtattttcca 120attaataaaa aatttagaga aacaattgta gcaccaacaa aaggtgtgac gacagaacaa 180tctcagaaat tgcaagttaa aattgttcca actttgacac aagatttaga aaatagtttt 240actgctagat ttactattgc tgttggcgat ggtcgggttt tggatatggg aagtacgtat 300tttgatatta ggggtaacat tgatcgggga ccttcattta agccatatgg tggtacagca 360tataatcctc tagctccaag gtcagctcaa tttaataata ttaaaactgt gggtggtaaa 420acatatttga ctgctcaagc tactaaattt ttttcaacat ctggaaatgg ttgtgcagct 480gctaatactg aagcaagttc atttacaaat ttagttcctt cacctaatac tggttcagca 540gaaagttctt ttgatcctac aacagaggga gctagttgca gagctataac acttggcagt 600tctgtaacag atgcaacttg ttatggagct tatacaccta ttcaaaatgc taatggttca 660attttacctc catctgttac gcctgataaa aaatttgccg atgctggtaa atctggcagt 720gttacatgta ctgctgctat ttgttgtgat aatgttactg tacaatatcc agatactaga 780atagttgctt atgactctac tgataaaata gcaactagaa tgggtaacag aattaattat 840attggattta gagataattt tataggtttg atgtattatg ataatggtgc acatagtggt 900tctttggcta cagaaacagg agatataaat ttggtagaac aattgcaaga tagaaataca 960gaaattagtt atcaatatat gttagcggat ttgatgagta ggaatcatta ttatagtcag 1020tggaatcaag ctgtagatga ttatgattta aatgttagag tacttacaaa tattggttat 1080gaagagggtc ctccaggtta ctgttatcca agcacaggca tgggcaacta tcctaatact 1140gtcatgtcgg ttgggacatt agtggataat aatggtacaa ctgctacaac aacgtcaaat 1200actgtagctg tgatgggttt tggcagtgtt cctactatgg aaattaacgt tcaagcttat 1260ttgcaaaaat gttggatgta tgctaacatt gcagaatatt tacctgataa gtataaaaaa 1320gctattcaag gtactagtga aactgatcca acaacttata gttatatgaa tagtaggctt 1380cctaatgtga atatggctga tctctttaca catattggcg ggcgttatag tttggatgta 1440atggataatg ttaatccttt taatcatcat agaaatagag gtttgcaata tagaagtcaa 1500attttgggta atggtagaaa tgtccgtttt catattcagg tacctcagaa attttttgct 1560attaagaatc tattgttact tcctggaact tatagttatg aatggtggtt caggaaagat 1620ccaaacttag tactacagtc tacgttggga aatgatttaa gaaaagatgg agcaagcatt 1680cagtttagca gtattagtct ttatgcgagt ttttttccta tggatcacgc tacttgtagt 1740gagcttattt taatgcttag aaacgatcaa aatgatcaaa cttttatgga ttatatgggt 1800gcaaagaata atttgtattt agttcctgct aatcaaacta atgttcagat tgaaatacct 1860tctagagctt ggacagcatt tagaggctgg agttttaacc gaattaaaac tgctgagaca 1920ccagctgtgt ggtctactta tgatcttaat tttaaatatt ctggctcaat accttatcta 1980gatggtacat tttatctttc tcacactttt aactctatgt ctattttgtt tgattcagca 2040ataacatggc caggtaatga tagaatgtta gttccgaatt tttttgaaat aaaaagagag 2100atagatacgg agggatacac tactagtcag tctaatatga ctaaagattg gtatttgatt 2160caaatgtctg caaattataa ccaggggtat cacggttata gttttccagc agataaagta 2220tacagacagt atgattttat gtcaaatttt gattctatgt ctgttcaagt accccggtca 2280ggtctggcat ttttgtttaa tgaaaattat aacttgatag taaataattc aggatttttg 2340cccagtagga cggctccaat tgctggagtt aatgaaggcc atccttatcc agcaaactgg 2400ccagcgccat taataggtaa tagtcctgat agtgttgtta cagttaggaa atttttatgt 2460gataagtatt tatggacaat acctttttca agcaatttta tgaatatggg tgaattgact 2520gaccttggac agagtttgct gtatactgag tctgcacata gtttgcaaat aacatttaat 2580gttgatccaa tgcctgagcc tacgtacatt tacttacttt atagtgtttt tgattgtgtt 2640agggtcaatc aacctaacaa aaattactta tctgcagctt atttcagaac tccttttgct 2700actggaactg cttcagta 2718 15 906 PRT hemorrhagic enteritis virus 15Met Asp Ile Ser Asn Ala Thr Pro Lys Leu Asp Ile Phe His Ile Ala1 5 10 15Gly Pro Asp Ala Ser Glu Tyr Leu Ser Glu Asn Leu Val Asn Phe Ile 20 25 30Ser Ser Thr Glu Ser Tyr Phe Pro Ile Asn Lys Lys Phe Arg Glu Thr 35 40 45Ile Val Ala Pro Thr Lys Gly Val Thr Thr Glu Gln Ser Gln Lys Leu 50 55 60Gln Val Lys Ile Val Pro Thr Leu Thr Gln Asp Leu Glu Asn Ser Phe65 70 75 80Thr Ala Arg Phe Thr Ile Ala Val Gly Asp Gly Arg Val Leu Asp Met 85 90 95Gly Ser Thr Tyr Phe Asp Ile Arg Gly Asn Ile Asp Arg Gly Pro Ser 100 105 110Phe Lys Pro Tyr Gly Gly Thr Ala Tyr Asn Pro Leu Ala Pro Arg Ser 115 120 125Ala Gln Phe Asn Asn Ile Lys Thr Val Gly Gly Lys Thr Tyr Leu Thr 130 135 140Ala Gln Ala Thr Lys Phe Phe Ser Thr Ser Gly Asn Gly Cys Ala Ala145 150 155 160Ala Asn Thr Glu Ala Ser Ser Phe Thr Asn Leu Val Pro Ser Pro Asn 165 170 175Thr Gly Ser Ala Glu Ser Ser Phe Asp Pro Thr Thr Glu Gly Ala Ser 180 185 190Cys Arg Ala Ile Thr Leu Gly Ser Ser Val Thr Asp Ala Thr Cys Tyr 195 200 205Gly Ala Tyr Thr Pro Ile Gln Asn Ala Asn Gly Ser Ile Leu Pro Pro 210 215 220Ser Val Thr Pro Asp Lys Lys Phe Ala Asp Ala Gly Lys Ser Gly Ser225 230 235 240Val Thr Cys Thr Ala Ala Ile Cys Cys Asp Asn Val Thr Val Gln Tyr 245 250 255Pro Asp Thr Arg Ile Val Ala Tyr Asp Ser Thr Asp Lys Ile Ala Thr 260 265 270Arg Met Gly Asn Arg Ile Asn Tyr Ile Gly Phe Arg Asp Asn Phe Ile 275 280 285Gly Leu Met Tyr Tyr Asp Asn Gly Ala His Ser Gly Ser Leu Ala Thr 290 295 300Glu Thr Gly Asp Ile Asn Leu Val Glu Gln Leu Gln Asp Arg Asn Thr305 310 315 320Glu Ile Ser Tyr Gln Tyr Met Leu Ala Asp Leu Met Ser Arg Asn His 325 330 335Tyr Tyr Ser Gln Trp Asn Gln Ala Val Asp Asp Tyr Asp Leu Asn Val 340 345 350Arg Val Leu Thr Asn Ile Gly Tyr Glu Glu Gly Pro Pro Gly Tyr Cys 355 360 365Tyr Pro Ser Thr Gly Met Gly Asn Tyr Pro Asn Thr Val Met Ser Val 370 375 380Gly Thr Leu Val Asp Asn Asn Gly Thr Thr Ala Thr Thr Thr Ser Asn385 390 395 400Thr Val Ala Val Met Gly Phe Gly Ser Val Pro Thr Met Glu Ile Asn 405 410 415Val Gln Ala Tyr Leu Gln Lys Cys Trp Met Tyr Ala Asn Ile Ala Glu 420 425 430Tyr Leu Pro Asp Lys Tyr Lys Lys Ala Ile Gln Gly Thr Ser Glu Thr 435 440 445Asp Pro Thr Thr Tyr Ser Tyr Met Asn Ser Arg Leu Pro Asn Val Asn 450 455 460Met Ala Asp Leu Phe Thr His Ile Gly Gly Arg Tyr Ser Leu Asp Val465 470 475 480Met Asp Asn Val Asn Pro Phe Asn His His Arg Asn Arg Gly Leu Gln 485 490 495Tyr Arg Ser Gln Ile Leu Gly Asn Gly Arg Asn Val Arg Phe His Ile 500 505 510Gln Val Pro Gln Lys Phe Phe Ala Ile Lys Asn Leu Leu Leu Leu Pro 515 520 525Gly Thr Tyr Ser Tyr Glu Trp Trp Phe Arg Lys Asp Pro Asn Leu Val 530 535 540Leu Gln Ser Thr Leu Gly Asn Asp Leu Arg Lys Asp Gly Ala Ser Ile545 550 555 560Gln Phe Ser Ser Ile Ser Leu Tyr Ala Ser Phe Phe Pro Met Asp His 565 570 575Ala Thr Cys Ser Glu Leu Ile Leu Met Leu Arg Asn Asp Gln Asn Asp 580 585 590Gln Thr Phe Met Asp Tyr Met Gly Ala Lys Asn Asn Leu Tyr Leu Val 595 600 605Pro Ala Asn Gln Thr Asn Val Gln Ile Glu Ile Pro Ser Arg Ala Trp 610 615 620Thr Ala Phe Arg Gly Trp Ser Phe Asn Arg Ile Lys Thr Ala Glu Thr625 630 635 640Pro Ala Val Trp Ser Thr Tyr Asp Leu Asn Phe Lys Tyr Ser Gly Ser 645 650 655Ile Pro Tyr Leu Asp Gly Thr Phe Tyr Leu Ser His Thr Phe Asn Ser 660 665 670Met Ser Ile Leu Phe Asp Ser Ala Ile Thr Trp Pro Gly Asn Asp Arg 675 680 685Met Leu Val Pro Asn Phe Phe Glu Ile Lys Arg Glu Ile Asp Thr Glu 690 695 700Gly Tyr Thr Thr Ser Gln Ser Asn Met Thr Lys Asp Trp Tyr Leu Ile705 710 715 720Gln Met Ser Ala Asn Tyr Asn Gln Gly Tyr His Gly Tyr Ser Phe Pro 725 730 735Ala Asp Lys Val Tyr Arg Gln Tyr Asp Phe Met Ser Asn Phe Asp Ser 740 745 750Met Ser Val Gln Val Pro Arg Ser Gly Leu Ala Phe Leu Phe Asn Glu 755 760 765Asn Tyr Asn Leu Ile Val Asn Asn Ser Gly Phe Leu Pro Ser Arg Thr 770 775 780Ala Pro Ile Ala Gly Val Asn Glu Gly His Pro Tyr Pro Ala Asn Trp785 790 795 800Pro Ala Pro Leu Ile Gly Asn Ser Pro Asp Ser Val Val Thr Val Arg 805 810 815Lys Phe Leu Cys Asp Lys Tyr Leu Trp Thr Ile Pro Phe Ser Ser Asn 820 825 830Phe Met Asn Met Gly Glu Leu Thr Asp Leu Gly Gln Ser Leu Leu Tyr 835 840 845Thr Glu Ser Ala His Ser Leu Gln Ile Thr Phe Asn Val Asp Pro Met 850 855 860Pro Glu Pro Thr Tyr Ile Tyr Leu Leu Tyr Ser Val Phe Asp Cys Val865 870 875 880Arg Val Asn Gln Pro Asn Lys Asn Tyr Leu Ser Ala Ala Tyr Phe Arg 885 890 895Thr Pro Phe Ala Thr Gly Thr Ala Ser Val 900 905 16 644 DNA hemorrhagic enteritis virus 16atggctggaa cttcaagttc agaattgatt acattagtac gttctttagg tttgggttct 60tattttttag gagtttatga taaacacttt cctggttttt taaatgatcg cagattagca 120tatgctattg taaatacggg tgattatatg tctggaggtt tacattggat agcttttgct 180tacgacccta atggtcgaaa attttacatt tttgatccat ttggttggtc aaaaaaggag 240ctttggaagt tttacaagtt tcagtatgat agaattgtta gaagaacagc gttacagaat 300ggtagatgta ttaaattagt tagatctgta gatactgttc agtgtccttg ttctgctgca 360tgtggtttgt attgtgtgtt atttttagct tcattttatt attttagaaa ttctcctatg 420tataataatc ctattattga tgttgttact ggcgtgccgc atagtaagat gaaatcttct 480tacggcatag ctatattaca ttgtaatcaa gaaagattgt ataactggct gtattacaac 540tctgtatatt ttcgagataa tgaattggaa ataaagagaa atacaagaat aaattctatt 600ttagttcatt atctctttat tgtattgttt ttatttgcgc gtta 644 17 214 PRT hemorrhagic enteritis virus 17Met Ala Gly Thr Ser Ser Ser Glu Leu Ile Thr Leu Val Arg Ser Leu1 5 10 15Gly Leu Gly Ser Tyr Phe Leu Gly Val Tyr Asp Lys His Phe Pro Gly 20 25 30Phe Leu Asn Asp Arg Arg Leu Ala Tyr Ala Ile Val Asn Thr Gly Asp 35 40 45Tyr Met Ser Gly Gly Leu His Trp Ile Ala Phe Ala Tyr Asp Pro Asn 50 55 60Gly Arg Lys Phe Tyr Ile Phe Asp Pro Phe Gly Trp Ser Lys Lys Glu65 70 75 80Leu Trp Lys Phe Tyr Lys Phe Gln Tyr Asp Arg Ile Val Arg Arg Thr 85 90 95Ala Leu Gln Asn Gly Arg Cys Ile Lys Leu Val Arg Ser Val Asp Thr 100 105 110Val Gln Cys Pro Cys Ser Ala Ala Cys Gly Leu Tyr Cys Val Leu Phe 115 120 125Leu Ala Ser Phe Tyr Tyr Phe Arg Asn Ser Pro Met Tyr Asn Asn Pro 130 135 140Ile Ile Asp Val Val Thr Gly Val Pro His Ser Lys Met Lys Ser Ser145 150 155 160Tyr Gly Ile Ala Ile Leu His Cys Asn Gln Glu Arg Leu Tyr Asn Trp 165 170 175Leu Tyr Tyr Asn Ser Val Tyr Phe Arg Asp Asn Glu Leu Glu Ile Lys 180 185 190Arg Asn Thr Arg Ile Asn Ser Ile Leu Val His Tyr Leu Phe Ile Val 195 200 205Leu Phe Leu Phe Ala Arg 210 18 2040 DNA hemorrhagic enteritis virus 18atgatctata aaagaggaaa agagagagga aattctaaaa ttataatggc ttcgtctgag 60gaggtcgtag actctgcagc gcaagaattc aatgaaccct tcccgccagc accagaaaca 120ttaccagatt cagaagttga tatagaactt atgaatcgtg acttgggtga gtttgaaaca 180aattctttta gcatccactt aaggagacaa gcacaattgt gcaaattggc tttacaagct 240aaattcaaat atttaccaga atctgtagct gaaattggag atgcattcga atcattcatt 300tttaatccaa ttactgaatc tgaccgaaaa caacaagagc ctagactcaa tttttaccct 360ccatttgctg tgccagaacg aacagcaact tacaatagct tttttcaaat tatgtctcta 420ccatttagct gcttagctaa cagatcaggt agtaaaaaat ataagactct aaaatcaatt 480acaaaatttg aagtcttacc caagtttgaa tcagatatgt ttgtgatttc agactgtctt 540gggtccgaag tctcagcaac agattctctg ccaaggaaaa caaggttggt taatttacaa 600tctgataaca taagattaat gtccatgaaa gaaaaactga agcatgtaac tcaatttgct 660tatccagcct tgaacattcc tccaaaaatt tataaaactc taattgagac actatataaa 720cctattcaac agggagagga tgatgaatct gattatgtgt tttcagatga tgatgttaga 780caagtcttta tttcaaattt agaggatttt gaaaaattta ctgatggaga gataggagaa 840ttaacaaatt gtttcagaaa aaacttgctt caggcaatac agtatgtgct acctttaaaa 900cttatgcaag gtacttttag acatccgtgc tttgtaaaga aattacaaga gatgttacat 960tatacttttc atcatggcta tatcaagtta attagttcta ttacgggcca caatttgagt 1020aaatatataa cttttcactg catgacatat gagaataaca ataacaatcc aaatcttcac 1080acaacattag atttgaatga tggtgaagat tatatggttg atacaatttt tttatacttg 1140ataatgactt ggcagactgc aatgggtgtg tggcaacaaa atatcaatga gaagaattta 1200gctagtatga aagatttttt aactaaaaac ggaccaaaat tgattttgtg tcgtgattca 1260gatagcatgg ctgatatgct agcagattgg ataacagatg gcggagtctt gcttcagatt 1320tttagggatg ctttaccaga ttttatgtca cagactcaat tgaataactt tagaacattt 1380attttagcga gaagtaatat agtgagctgt atggtttcaa cagtagttaa agattttgta 1440ccattagatt ttaaagaatc tccaccacaa ttgtggccac atgtttactg cttgagactg 1500tcttattttt tctacaatca tggagattat caacaaattt tttattggga cgataataaa 1560cctacagaaa atgaaatttt ttgttattgc aatctttgtg ctcctcatag aacaccaatg 1620ctgaacacag ctttacacaa tgaaatttta gcaattgggt cgtttgactt ttttgttcca 1680agtagtgatg gtaaaggtgg agaaagagtt acattaactc cgggattatg ggctaataaa 1740tttttgaatc attttgtaag ttctgaatat tttccatttg aagttaaaaa atatgtagac 1800catccagaat gtttcaaaat acctcctaca gcatgtgtaa ttactaagcc tgagatttta 1860agtagtttga aagagataaa gaagaggaga gaaaagtttt taattgaaaa aggttctggt 1920atttatttgg atcctcaaac gggagataac ttaagtgatg ctaaaattgt ttcacagccc 1980agaagaggca gcagtggcgg aaaaacagaa aaagaagaaa cggcaaagaa gaatcccagg 2040 19 680 PRT hemorrhagic enteritis virus 19Met Ile Tyr Lys Arg Gly Lys Glu Arg Gly Asn Ser Lys Ile Ile Met1 5 10 15Ala Ser Ser Glu Glu Val Val Asp Ser Ala Ala Gln Glu Phe Asn Glu 20 25 30Pro Phe Pro Pro Ala Pro Glu Thr Leu Pro Asp Ser Glu Val Asp Ile 35 40 45Glu Leu Met Asn Arg Asp Leu Gly Glu Phe Glu Thr Asn Ser Phe Ser 50 55 60Ile His Leu Arg Arg Gln Ala Gln Leu Cys Lys Leu Ala Leu Gln Ala65 70 75 80Lys Phe Lys Tyr Leu Pro Glu Ser Val Ala Glu Ile Gly Asp Ala Phe 85 90 95Glu Ser Phe Ile Phe Asn Pro Ile Thr Glu Ser Asp Arg Lys Gln Gln 100 105 110Glu Pro Arg Leu Asn Phe Tyr Pro Pro Phe Ala Val Pro Glu Arg Thr 115 120 125Ala Thr Tyr Asn Ser Phe Phe Gln Ile Met Ser Leu Pro Phe Ser Cys 130 135 140Leu Ala Asn Arg Ser Gly Ser Lys Lys Tyr Lys Thr Leu Lys Ser Ile145 150 155 160Thr Lys Phe Glu Val Leu Pro Lys Phe Glu Ser Asp Met Phe Val Ile 165 170 175Ser Asp Cys Leu Gly Ser Glu Val Ser Ala Thr Asp Ser Leu Pro Arg 180 185 190Lys Thr Arg Leu Val Asn Leu Gln Ser Asp Asn Ile Arg Leu Met Ser 195 200 205Met Lys Glu Lys Leu Lys His Val Thr Gln Phe Ala Tyr Pro Ala Leu 210 215 220Asn Ile Pro Pro Lys Ile Tyr Lys Thr Leu Ile Glu Thr Leu Tyr Lys225 230 235 240Pro Ile Gln Gln Gly Glu Asp Asp Glu Ser Asp Tyr Val Phe Ser Asp 245 250 255Asp Asp Val Arg Gln Val Phe Ile Ser Asn Leu Glu Asp Phe Glu Lys 260 265 270Phe Thr Asp Gly Glu Ile Gly Glu Leu Thr Asn Cys Phe Arg Lys Asn 275 280 285Leu Leu Gln Ala Ile Gln Tyr Val Leu Pro Leu Lys Leu Met Gln Gly 290 295 300Thr Phe Arg His Pro Cys Phe Val Lys Lys Leu Gln Glu Met Leu His305 310 315 320Tyr Thr Phe His His Gly Tyr Ile Lys Leu Ile Ser Ser Ile Thr Gly 325 330 335His Asn Leu Ser Lys Tyr Ile Thr Phe His Cys Met Thr Tyr Glu Asn 340 345 350Asn Asn Asn Asn Pro Asn Leu His Thr Thr Leu Asp Leu Asn Asp Gly 355 360 365Glu Asp Tyr Met Val Asp Thr Ile Phe Leu Tyr Leu Ile Met Thr Trp 370 375 380Gln Thr Ala Met Gly Val Trp Gln Gln Asn Ile Asn Glu Lys Asn Leu385 390 395 400Ala Ser Met Lys Asp Phe Leu Thr Lys Asn Gly Pro Lys Leu Ile Leu 405 410 415Cys Arg Asp Ser Asp Ser Met Ala Asp Met Leu Ala Asp Trp Ile Thr 420 425 430Asp Gly Gly Val Leu Leu Gln Ile Phe Arg Asp Ala Leu Pro Asp Phe 435 440 445Met Ser Gln Thr Gln Leu Asn Asn Phe Arg Thr Phe Ile Leu Ala Arg 450 455 460Ser Asn Ile Val Ser Cys Met Val Ser Thr Val Val Lys Asp Phe Val465 470 475 480Pro Leu Asp Phe Lys Glu Ser Pro Pro Gln Leu Trp Pro His Val Tyr 485 490 495Cys Leu Arg Leu Ser Tyr Phe Phe Tyr Asn His Gly Asp Tyr Gln Gln 500 505 510Ile Phe Tyr Trp Asp Asp Asn Lys Pro Thr Glu Asn Glu Ile Phe Cys 515 520 525Tyr Cys Asn Leu Cys Ala Pro His Arg Thr Pro Met Leu Asn Thr Ala 530 535 540Leu His Asn Glu Ile Leu Ala Ile Gly Ser Phe Asp Phe Phe Val Pro545 550 555 560Ser Ser Asp Gly Lys Gly Gly Glu Arg Val Thr Leu Thr Pro Gly Leu 565 570 575Trp Ala Asn Lys Phe Leu Asn His Phe Val Ser Ser Glu Tyr Phe Pro 580 585 590Phe Glu Val Lys Lys Tyr Val Asp His Pro Glu Cys Phe Lys Ile Pro 595 600 605Pro Thr Ala Cys Val Ile Thr Lys Pro Glu Ile Leu Ser Ser Leu Lys 610 615 620Glu Ile Lys Lys Arg Arg Glu Lys Phe Leu Ile Glu Lys Gly Ser Gly625 630 635 640Ile Tyr Leu Asp Pro Gln Thr Gly Asp Asn Leu Ser Asp Ala Lys Ile 645 650 655Val Ser Gln Pro Arg Arg Gly Ser Ser Gly Gly Lys Thr Glu Lys Glu 660 665 670Glu Thr Ala Lys Lys Asn Pro Arg 675 680 20 600 DNA hemorrhagic enteritis virus 20atggatcccg ttccgttaga atatatttgg cagtacaacc ctgtgactgg tagagttgga 60ggagcaaatc agaattacgg acagaggatt aatgttttgc atacaaatcg ttacctctac 120aacagaatgc agaatgtgca gaaaaaaagc aacgaaagag ccactgaaag aggattacta 180tctctgaagg gaggaagcac attgccgact attgcagaag atgaacctgc ccagttgaac 240tcggctatag ttcgcatggc aggattgaat gacttgaaca cggttcaagc accggattca 300tctaatctgc aaaacctggc caacattgca cttgaagctg ctgaacacaa caaagctacc 360tcatctctcc taacaacgaa gaagtttgtt gaagagtttc ctcctgttgt ttacgaaaat 420cctttttctg gctctaattt tgcttatgaa tttaatcctc tgtattcacc gtccgggaat 480gagttctcca atcctccatt taaattcacc ggaggtgcta ttagcctcac aggacaacac 540ccagtcctct cgggaggtgc tgttatctta tcaggacaga atccggtact ggacaaagca 600 21 200 PRT hemorrhagic enteritis virus 21Met Asp Pro Val Pro Leu Glu Tyr Ile Trp Gln Tyr Asn Pro Val Thr1 5 10 15Gly Arg Val Gly Gly Ala Asn Gln Asn Tyr Gly Gln Arg Ile Asn Val 20 25 30Leu His Thr Asn Arg Tyr Leu Tyr Asn Arg Met Gln Asn Val Gln Lys 35 40 45Lys Ser Asn Glu Arg Ala Thr Glu Arg Gly Leu Leu Ser Leu Lys Gly 50 55 60Gly Ser Thr Leu Pro Thr Ile Ala Glu Asp Glu Pro Ala Gln Leu Asn65 70 75 80Ser Ala Ile Val Arg Met Ala Gly Leu Asn Asp Leu Asn Thr Val Gln 85 90 95Ala Pro Asp Ser Ser Asn Leu Gln Asn Leu Ala Asn Ile Ala Leu Glu 100 105 110Ala Ala Glu His Asn Lys Ala Thr Ser Ser Leu Leu Thr Thr Lys Lys 115 120 125Phe Val Glu Glu Phe Pro Pro Val Val Tyr Glu Asn Pro Phe Ser Gly 130 135 140Ser Asn Phe Ala Tyr Glu Phe Asn Pro Leu Tyr Ser Pro Ser Gly Asn145 150 155 160Glu Phe Ser Asn Pro Pro Phe Lys Phe Thr Gly Gly Ala Ile Ser Leu 165 170 175Thr Gly Gln His Pro Val Leu Ser Gly Gly Ala Val Ile Leu Ser Gly 180 185 190Gln Asn Pro Val Leu Asp Lys Ala 195 200 22 1362 DNA hemorrhagic enteritis virus 22atggctactc ctggaaagcg ttctgcagag gaaccagatc aacagacctt gaaaaagtca 60aaacaatctg accaaagtca gggtttaaat ctagcatatc cttttgataa aataacagaa 120tttgaagcaa cacctccctt tattcatgtt gggcaaggct tagacatatc agatttatcg 180ttaaatatga gaattggcaa aggattaaag tttgaaaatg gtaatctagt tgtatcagat 240caacagtata atgttacacc acctttaatt gcagatcagt caacattagg tttaaagtat 300aatccggatg ttctttcttt aacacattca ggtgctttaa ctttgccaac tattcaacat 360cccctccagg cttcagctgg aaaatttgaa cttgctttgt catcaggttt aaaatctgat 420gatcaaggtt taactttaga tttggatcct gtattttcta cagaatcatc aaaatttttg 480cttaattgtt cattgccgtt agataagaat agtgacaagt taacgttaaa atttggtaat 540ggtcttggat tgaataatga ccagctagag aatactatga cttataatct tcctttaaaa 600cgtgatggaa ctaatgttag tctttcattt ggaactaatt tcaaaatatt gaatgagatg 660ttagatttaa atcttgtggc acctatgtct aattcagcag gaggattagc attgcaattt 720aaaagccctt tgtcagcaga tgatggtatt ttatcaatta aaacagatac atctttgggt 780ataacaggaa ataaattagg aataagattg gcccctaaca gtggtctgca aataacacca 840aatggtctag cagttagtgt taatgctgtg caaattctaa gtagtccttt aattactgca 900gcgtctatag gcccaccaac aacaatggtt actggaacag tgtcaccggg cagagcaaca 960aatggtcaat ttgtaaccaa aactgctaaa gttttacgtt ataaatttgt gagatgggat 1020gctctgttaa tcatacagtt tatagataac ataggtgtaa tagaaaaccc taccttttat 1080cgtaacaaaa gtattgaatt aagatctgct gatttcttga gtcctacgtt aaataataca 1140tatatagtgc cattgaatgg aggggtaagg gtagaatcac ctactattcc tgtacaatta 1200gaagttatac ttgaaaacaa ttcctctttc attcaagtag ggtttgttag gttaacagtt 1260aagaatggta accctcatat gattattcag tgtaatcctg tacctgggaa tattaaaatg 1320ataaagataa aatctgtaat gctttttact tgtttgatag gc 1362 23 454 PRT hemorrhagic enteritis virus 23Met Ala Thr Pro Gly Lys Arg Ser Ala Glu Glu Pro Asp Gln Gln Thr1 5 10 15Leu Lys Lys Ser Lys Gln Ser Asp Gln Ser Gln Gly Leu Asn Leu Ala 20 25 30Tyr Pro Phe Asp Lys Ile Thr Glu Phe Glu Ala Thr Pro Pro Phe Ile 35 40 45His Val Gly Gln Gly Leu Asp Ile Ser Asp Leu Ser Leu Asn Met Arg 50 55 60Ile Gly Lys Gly Leu Lys Phe Glu Asn Gly Asn Leu Val Val Ser Asp65 70 75 80Gln Gln Tyr Asn Val Thr Pro Pro Leu Ile Ala Asp Gln Ser Thr Leu 85 90 95Gly Leu Lys Tyr Asn Pro Asp Val Leu Ser Leu Thr His Ser Gly Ala 100 105 110Leu Thr Leu Pro Thr Ile Gln His Pro Leu Gln Ala Ser Ala Gly Lys 115 120 125Phe Glu Leu Ala Leu Ser Ser Gly Leu Lys Ser Asp Asp Gln Gly Leu 130 135 140Thr Leu Asp Leu Asp Pro Val Phe Ser Thr Glu Ser Ser Lys Phe Leu145 150 155 160Leu Asn Cys Ser Leu Pro Leu Asp Lys Asn Ser Asp Lys Leu Thr Leu 165 170 175Lys Phe Gly Asn Gly Leu Gly Leu Asn Asn Asp Gln Leu Glu Asn Thr 180 185 190Met Thr Tyr Asn Leu Pro Leu Lys Arg Asp Gly Thr Asn Val Ser Leu 195 200 205Ser Phe Gly Thr Asn Phe Lys Ile Leu Asn Glu Met Leu Asp Leu Asn 210 215 220Leu Val Ala Pro Met Ser Asn Ser Ala Gly Gly Leu Ala Leu Gln Phe225 230 235 240Lys Ser Pro Leu Ser Ala Asp Asp Gly Ile Leu Ser Ile Lys Thr Asp 245 250 255Thr Ser Leu Gly Ile Thr Gly Asn Lys Leu Gly Ile Arg Leu Ala Pro 260 265 270Asn Ser Gly Leu Gln Ile Thr Pro Asn Gly Leu Ala Val Ser Val Asn 275 280 285Ala Val Gln Ile Leu Ser Ser Pro Leu Ile Thr Ala Ala Ser Ile Gly 290 295 300Pro Pro Thr Thr Met Val Thr Gly Thr Val Ser Pro Gly Arg Ala Thr305 310 315 320Asn Gly Gln Phe Val Thr Lys Thr Ala Lys Val Leu Arg Tyr Lys Phe 325 330 335Val Arg Trp Asp Ala Leu Leu Ile Ile Gln Phe Ile Asp Asn Ile Gly 340 345 350Val Ile Glu Asn Pro Thr Phe Tyr Arg Asn Lys Ser Ile Glu Leu Arg 355 360 365Ser Ala Asp Phe Leu Ser Pro Thr Leu Asn Asn Thr Tyr Ile Val Pro 370 375 380Leu Asn Gly Gly Val Arg Val Glu Ser Pro Thr Ile Pro Val Gln Leu385 390 395 400Glu Val Ile Leu Glu Asn Asn Ser Ser Phe Ile Gln Val Gly Phe Val 405 410 415Arg Leu Thr Val Lys Asn Gly Asn Pro His Met Ile Ile Gln Cys Asn 420 425 430Pro Val Pro Gly Asn Ile Lys Met Ile Lys Ile Lys Ser Val Met Leu 435 440 445Phe Thr Cys Leu Ile Gly 450 24 1101 DNA hemorrhagic enteritis virus 24atgaataatg aagttgaaga attctacgat gtagttggac aatggaaaac tgctgtagac 60agtattaact caagcatact accaggatgt gatttgccgc catttaaaga atttgaaagc 120tataactcta atatagactt gagaagcaat atgaggaaat acaacgaaat taacaacatt 180aacaagatgt atctaactaa aaactctgaa ctgccttcta tcaatatgaa ctctgatcca 240cttattagtt tagtaatcgg acctacaggt tgcgggaaaa gccaattaat tagaaattta 300ctgggattca aaaaaataca accaatgcca gaaacgatca tcttcataac tcctacaaaa 360ggaactattt catatgacga agtaatatta tggaaaactc aactacaaga aggaaattat 420tctgctcaag ataataccct ttacccaacc acaaaggtat tcaccattaa ttttctagaa 480tgtgcttttg atgatgtcat taccccagag aatctagatg ttaacaacga aaattctatc 540tttaatattc ataccaaaaa agggccagta tgcgtaattc tagatgaatg tatgcagaaa 600ttaatacaga aaccaaatat aagcccatta tattgtagtt taccatcaaa gctatctagt 660agatatggtc atgcattcta tatgttcgtt gttttacata atgtaaatcc gttatcagga 720aacggtaata atattatgga cttgaagact caagccaaat tacacatttt aagcacaaaa 780aaccaaccac tacaattatc taattttgta cataatcgat caggaggaat ggactctaac 840gtacgaacga tactattaaa cagtattgta tctgaaaaaa atccgaaata ttcttttgta 900atgtttaata cttgtccaac aagggaatca tttcaatggt cagcaatact cgagggaggg 960aaaagtatta tacctttatg tttagatatg caaagtttac ttctagactc tgtaaataaa 1020atctgtaaca ttcacttatg taaattaaaa aataagaaaa ggtacttgaa agaaaaacaa 1080aaacgcatgt atgatgaaat t 1101 25 367 PRT hemorrhagic enteritis virus 25Met Asn Asn Glu Val Glu Glu Phe Tyr Asp Val Val Gly Gln Trp Lys1 5 10 15Thr Ala Val Asp Ser Ile Asn Ser Ser Ile Leu Pro Gly Cys Asp Leu 20 25 30Pro Pro Phe Lys Glu Phe Glu Ser Tyr Asn Ser Asn Ile Asp Leu Arg 35 40 45Ser Asn Met Arg Lys Tyr Asn Glu Ile Asn Asn Ile Asn Lys Met Tyr 50 55 60Leu Thr Lys Asn Ser Glu Leu Pro Ser Ile Asn Met Asn Ser Asp Pro65 70 75 80Leu Ile Ser Leu Val Ile Gly Pro Thr Gly Cys Gly Lys Ser Gln Leu 85 90 95Ile Arg Asn Leu Leu Gly Phe Lys Lys Ile Gln Pro Met Pro Glu Thr 100 105 110Ile Ile Phe Ile Thr Pro Thr Lys Gly Thr Ile Ser Tyr Asp Glu Val 115 120 125Ile Leu Trp Lys Thr Gln Leu Gln Glu Gly Asn Tyr Ser Ala Gln Asp 130 135 140Asn Thr Leu Tyr Pro Thr Thr Lys Val Phe Thr Ile Asn Phe Leu Glu145 150 155 160Cys Ala Phe Asp Asp Val Ile Thr Pro Glu Asn Leu Asp Val Asn Asn 165 170 175Glu Asn Ser Ile Phe Asn Ile His Thr Lys Lys Gly Pro Val Cys Val 180 185 190Ile Leu Asp Glu Cys Met Gln Lys Leu Ile Gln Lys Pro Asn Ile Ser 195 200 205Pro Leu Tyr Cys Ser Leu Pro Ser Lys Leu Ser Ser Arg Tyr Gly His 210 215 220Ala Phe Tyr Met Phe Val Val Leu His Asn Val Asn Pro Leu Ser Gly225 230 235 240Asn Gly Asn Asn Ile Met Asp Leu Lys Thr Gln Ala Lys Leu His Ile 245 250 255Leu Ser Thr Lys Asn Gln Pro Leu Gln Leu Ser Asn Phe Val His Asn 260 265 270Arg Ser Gly Gly Met Asp Ser Asn Val Arg Thr Ile Leu Leu Asn Ser 275 280 285Ile Val Ser Glu Lys Asn Pro Lys Tyr Ser Phe Val Met Phe Asn Thr 290 295 300Cys Pro Thr Arg Glu Ser Phe Gln Trp Ser Ala Ile Leu Glu Gly Gly305 310 315 320Lys Ser Ile Ile Pro Leu Cys Leu Asp Met Gln Ser Leu Leu Leu Asp 325 330 335Ser Val Asn Lys Ile Cys Asn Ile His Leu Cys Lys Leu Lys Asn Lys 340 345 350Lys Arg Tyr Leu Lys Glu Lys Gln Lys Arg Met Tyr Asp Glu Ile 355 360 365 26 3336 DNA hemorrhagic enteritis virus 26ttcatcttcg aattcctgta gtaagatagg atttgttatt ctggggtttg ggtggtagag 60atcataagga attaaagtgt ttgtctttag aaagtaaagg gttggatcat tccacggccg 120tagttcgcgt attaattgta tttcatgaac agaaaagggg gagaattttc cgtaagactt 180acataacgtt cttttcaaag cagttctttc tgtttggaag atctcaccag attttaattc 240tgaagtgtgg tagttgaagc attcttccag gatttcaaaa gttatgtctg cggtacagtg 300accctttgct cgtaattttc ctgtttttga atttttgcag gtggtacacg tgatttcttt 360aagggcgtac agctttggag cgagaaaaat actttttgaa gaatatgcag gtgaattaca 420caaattacac caagtttcac attctactgc ccaaacaatt gaaggttttt taggatcaaa 480tgttagttgt gaatttttgt ttttaattct atgttgtcca tattttaaca tattttgatg 540tcctttttct gtgagaaata gactgtctgt gtcaccgtag attgatttaa tggttttgaa 600ttgaactggt atactatctt catcactgta taggatttcg cgccactcgc tcataaaggc 660tcttgtccac gctaatacga agcttgctag ttgagttgga tatcttttgt ttgtcggatg 720taaattcgtt gattttaaca tgtaaatagt gaggttttct gatgtgacgt ctaagatgtt 780gaatggtttg taggttccca cgtgagcagt cgaagttgaa gtttgtgagg ctgggctttc 840atccataaat tctaaactgt taaacggact ggtaagctca tcatccagtt ctaaatttct 900ctctgcactg tcagttcggt tttttaaggt aaatgttaaa ttttctatac taacatatgg 960aaggttgtaa gttggtagag tagtaatgct gtcaatggtt agttgtttgt tcaataattg 1020atttcttatt ttaggatttt cttgaatgtg gttttcgaaa attgtaatgt cattgctttc 1080tcttgtagca aaactaccgt atagtgcatt acttaggagt ttgctgattg ctcttttaac 1140aggatttttt tcaacggtgg ctttttcttt tgctagaata tttttagtaa catattctaa 1200gcagcaagtg ttccacgttg ggaatactgt gtttagcttg tggggaataa tgtttacttt 1260ccatcctcta ttatggagtg tgataatatc aattgaggta acaatttcat ttttcagtat 1320ttcgttcgtc caacataatt tgccgctttg tcttgaacat aatggcggta gaggatcaag 1380atgttctgga ggtggcggga tggcgtcaat cattactacc attggcttta tatcagtaaa 1440atatgatagt tttgttttag ttttttttaa tttttcgttt aactttgtaa tttcattgtt 1500tctttctttc tctcctattg gaaatccgta tggcattgga tgagttagag cactagcata 1560catgccgcaa atatcgtaaa caaaaatttt ttctttgaat attcctaagt gtgttgggta 1620acaacgtcca cctctaacgg actgacggat gaaactgtac atttcatcag atggtgctac 1680tatgtctggc agggtggagg cttttgttcc attttgtttg aagtgtaact gtctaaaaat 1740ggcgtgagaa ttagatgaaa tagtcggtct tttaaaaata ttaaaattgc attttaggtt 1800tagttcctct ttgatgaatg tatcgaaagt atttaggagg gtttttgtta gttcttccgt 1860tattttaacg tctttaatac aatagtcgat taattcttta acaatgtcat atttgacttc 1920ctttttttct aaccaaattt ctttttgttc gttatattct tctttgctag accaatattt 1980ttctgatgga aacgagtctg agtctgtaga taatgaattg gttgacatgt actcatttat 2040agctttgaac ggacaacaac ctttgtgaat ttttaagttg tatgctttgg ccgcttcttt 2100tagagaagtg tgtgttattt ggaaagtatc tctgaccata gacttgatat ataaattttt 2160tatactttct ggatgtggaa ttccttcttt aatctgtgaa agtatttcac ttttattatt 2220ttttttttct tctttagcta catagtagtc tgggttgggg aatttaattg taatatcgtt 2280aaaaagtatt ctgccttgtc ttggcataaa atttctttca actgtgaaaa ggggagtgat 2340gtctaggttt tcgtcttgaa ggatttgtgt tgctaataga atttcatcga atgactgaat 2400gttgtgtccg atgatgtaaa attctatgaa aattggttta actttcagac tcattatgag 2460gttagtatgt tcacttaaat tgatatttgt aattgattct aaattttgtg tttgagcgaa 2520atcttcgaga atttctcggt tttctggtgt taaaattaat tttgtgaagt aatctgtgag 2580tgcaaatagg atgttatttc ttaattttct gaactccgta ctaatgaagt ttttaacttt 2640agaaaaccag aagtacgtag aattttttgt ttgaatagat tttattttgg ctatttctgt 2700tttacagata ttgataagac aatcatctcc gaaaattgaa aagcatagta gaagaggatt 2760taataaaact ccagatgttt cagctagtgt aaatgtttct atatcgtaga taaggaagag 2820tttttttgtg ttttcatttt ctcctatagg ttgaaatggt atagtttccc agaattttct 2880tgtgtcattg tctacgctgt tgtagtagta agtagatctt aagtcattgc aggtatgaat 2940tgcggagtag attctaccac atgagctgca tttttgttgt tgagtccatg attttatcca 3000gcataatttt gacttatcat atatgaagtc taattcaata tattcatgcg atgttttgaa 3060ttcattttgg atcataacac atccagttaa ttttttccag atagttattg atttgaccgg 3120taagtttttt acattttcaa tatcgctgtt tgtgggcttg tatttactac aaatgaggat 3180atatgattgt gcatctttta ctttttttgc aagtttttta caattaaata aattatatgt 3240ccaagctatt tctattatag tggcttttat gtctttttta catatttgca acctgtatgg 3300aatttcgttt tgagtggtat aaatgtattt gctcat 3336 27 1112 PRT hemorrhagic enteritis virus 27Met Ser Lys Tyr Ile Tyr Thr Thr Gln Asn Glu Ile Pro Tyr Arg Leu1 5 10 15Gln Ile Cys Lys Lys Asp Ile Lys Ala Thr Ile Ile Glu Ile Ala Trp 20 25 30Thr Tyr Asn Leu Phe Asn Cys Lys Lys Leu Ala Lys Lys Val Lys Asp 35 40 45Ala Gln Ser Tyr Ile Leu Ile Cys Ser Lys Tyr Lys Pro Thr Asn Ser 50 55 60Asp Ile Glu Asn Val Lys Asn Leu Pro Val Lys Ser Ile Thr Ile Trp65 70 75 80Lys Lys Leu Thr Gly Cys Val Met Ile Gln Asn Glu Phe Lys Thr Ser 85 90 95His Glu Tyr Ile Glu Leu Asp Phe Ile Tyr Asp Lys Ser Lys Leu Cys 100 105 110Trp Ile Lys Ser Trp Thr Gln Gln Gln Lys Cys Ser Ser Cys Gly Arg 115 120 125Ile Tyr Ser Ala Ile His Thr Cys Asn Asp Leu Arg Ser Thr Tyr Tyr 130 135 140Tyr Asn Ser Val Asp Asn Asp Thr Arg Lys Phe Trp Glu Thr Ile Pro145 150 155 160Phe Gln Pro Ile Gly Glu Asn Glu Asn Thr Lys Lys Leu Phe Leu Ile 165 170 175Tyr Asp Ile Glu Thr Phe Thr Leu Ala Glu Thr Ser Gly Val Leu Leu 180 185 190Asn Pro Leu Leu Leu Cys Phe Ser Ile Phe Gly Asp Asp Cys Leu Ile 195 200 205Asn Ile Cys Lys Thr Glu Ile Ala Lys Ile Lys Ser Ile Gln Thr Lys 210 215 220Asn Ser Thr Tyr Phe Trp Phe Ser Lys Val Lys Asn Phe Ile Ser Thr225 230 235 240Glu Phe Arg Lys Leu Arg Asn Asn Ile Leu Phe Ala Leu Thr Asp Tyr 245 250 255Phe Thr Lys Leu Ile Leu Thr Pro Glu Asn Arg Glu Ile Leu Glu Asp 260 265 270Phe Ala Gln Thr Gln Asn Leu Glu Ser Ile Thr Asn Ile Asn Leu Ser 275 280 285Glu His Thr Asn Leu Ile Met Ser Leu Lys Val Lys Pro Ile Phe Ile 290 295 300Glu Phe Tyr Ile Ile Gly His Asn Ile Gln Ser Phe Asp Glu Ile Leu305 310 315 320Leu Ala Thr Gln Ile Leu Gln Asp Glu Asn Leu Asp Ile Thr Pro Leu 325 330 335Phe Thr Val Glu Arg Asn Phe Met Pro Arg Gln Gly Arg Ile Leu Phe 340 345 350Asn Asp Ile Thr Ile Lys Phe Pro Asn Pro Asp Tyr Tyr Val Ala Lys 355 360 365Glu Glu Lys Lys Asn Asn Lys Ser Glu Ile Leu Ser Gln Ile Lys Glu 370 375 380Gly Ile Pro His Pro Glu Ser Ile Lys Asn Leu Tyr Ile Lys Ser Met385 390 395 400Val Arg Asp Thr Phe Gln Ile Thr His Thr Ser Leu Lys Glu Ala Ala 405 410 415Lys Ala Tyr Asn Leu Lys Ile His Lys Gly Cys Cys Pro Phe Lys Ala 420 425 430Ile Asn Glu Tyr Met Ser Thr Asn Ser Leu Ser Thr Asp Ser Asp Ser 435 440 445Phe Pro Ser Glu Lys Tyr Trp Ser Ser Lys Glu Glu Tyr Asn Glu Gln 450 455 460Lys Glu Ile Trp Leu Glu Lys Lys Glu Val Lys Tyr Asp Ile Val Lys465 470 475 480Glu Leu Ile Asp Tyr Cys Ile Lys Asp Val Lys Ile Thr Glu Glu Leu 485 490 495Thr Lys Thr Leu Leu Asn Thr Phe Asp Thr Phe Ile Lys Glu Glu Leu 500 505 510Asn Leu Lys Cys Asn Phe Asn Ile Phe Lys Arg Pro Thr Ile Ser Ser 515 520 525Asn Ser His Ala Ile Phe Arg Gln Leu His Phe Lys Gln Asn Gly Thr 530 535 540Lys Ala Ser Thr Leu Pro Asp Ile Val Ala Pro Ser Asp Glu Met Tyr545 550 555 560Ser Phe Ile Arg Gln Ser Val Arg Gly Gly Arg Cys Tyr Pro Thr His 565 570 575Leu Gly Ile Phe Lys Glu Lys Ile Phe Val Tyr Asp Ile Cys Gly Met 580 585 590Tyr Ala Ser Ala Leu Thr His Pro Met Pro Tyr Gly Phe Pro Ile Gly 595 600 605Glu Lys Glu Arg Asn Asn Glu Ile Thr Lys Leu Asn Glu Lys Leu Lys 610 615 620Lys Thr Lys Thr Lys Leu Ser Tyr Phe Thr Asp Ile Lys Pro Met Val625 630 635 640Val Met Ile Asp Ala Ile Pro Pro Pro Pro Glu His Leu Asp Pro Leu 645 650 655Pro Pro Leu Cys Ser Arg Gln Ser Gly Lys Leu Cys Trp Thr Asn Glu 660 665 670Ile Leu Lys Asn Glu Ile Val Thr Ser Ile Asp Ile Ile Thr Leu His 675 680 685Asn Arg Gly Trp Lys Val Asn Ile Ile Pro His Lys Leu Asn Thr Val 690 695 700Phe Pro Thr Trp Asn Thr Cys Cys Leu Glu Tyr Val Thr Lys Asn Ile705 710 715 720Leu Ala Lys Glu Lys Ala Thr Val Glu Lys Asn Pro Val Lys Arg Ala 725 730 735Ile Ser Lys Leu Leu Ser Asn Ala Leu Tyr Gly Ser Phe Ala Thr Arg 740 745 750Glu Ser Asn Asp Ile Thr Ile Phe Glu Asn His Ile Gln Glu Asn Pro 755 760 765Lys Ile Arg Asn Gln Leu Leu Asn Lys Gln Leu Thr Ile Asp Ser Ile 770 775 780Thr Thr Leu Pro Thr Tyr Asn Leu Pro Tyr Val Ser Ile Glu Asn Leu785 790 795 800Thr Phe Thr Leu Lys Asn Arg Thr Asp Ser Ala Glu Arg Asn Leu Glu 805 810 815Leu Asp Asp Glu Leu Thr Ser Pro Phe Asn Ser Leu Glu Phe Met Asp 820 825 830Glu Ser Pro Ala Ser Gln Thr Ser Thr Ser Thr Ala His Val Gly Thr 835 840 845Tyr Lys Pro Phe Asn Ile Leu Asp Val Thr Ser Glu Asn Leu Thr Ile 850 855 860Tyr Met Leu Lys Ser Thr Asn Leu His Pro Thr Asn Lys Arg Tyr Pro865 870 875 880Thr Gln Leu Ala Ser Phe Val Leu Ala Trp Thr Arg Ala Phe Met Ser 885 890 895Glu Trp Arg Glu Ile Leu Tyr Ser Asp Glu Asp Ser Ile Pro Val Gln 900 905 910Phe Lys Thr Ile Lys Ser Ile Tyr Gly Asp Thr Asp Ser Leu Phe Leu 915 920 925Thr Glu Lys Gly His Gln Asn Met Leu Lys Tyr Gly Gln His Arg Ile 930 935 940Lys Asn Lys Asn Ser Gln Leu Thr Phe Asp Pro Lys Lys Pro Ser Ile945 950 955 960Val Trp Ala Val Glu Cys Glu Thr Trp Cys Asn Leu Cys Asn Ser Pro 965 970 975Ala Tyr Ser Ser Lys Ser Ile Phe Leu Ala Pro Lys Leu Tyr Ala Leu 980 985 990Lys Glu Ile Thr Cys Thr Thr Cys Lys Asn Ser Lys Thr Gly Lys Leu 995 1000 1005Arg Ala Lys Gly His Cys Thr Ala Asp Ile Thr Phe Glu Ile Leu 1010 1015 1020Glu Glu Cys Phe Asn Tyr His Thr Ser Glu Leu Lys Ser Gly Glu 1025 1030 1035Ile Phe Gln Thr Glu Arg Thr Ala Leu Lys Arg Thr Leu Cys Lys 1040 1045 1050Ser Tyr Gly Lys Phe Ser Pro Phe Ser Val His Glu Ile Gln Leu 1055 1060 1065Ile Arg Glu Leu Arg Pro Trp Asn Asp Pro Thr Leu Tyr Phe Leu 1070 1075 1080Lys Thr Asn Thr Leu Ile Pro Tyr Asp Leu Tyr His Pro Asn Pro 1085 1090 1095Arg Ile Thr Asn Pro Ile Leu Leu Gln Glu Phe Glu Asp Glu 1100 1105 1110 28 1791 DNA hemorrhagic enteritis virus 28atgtctttct ttcagattaa caattatgcc cgcctaacta accaagaaga agacaccatc 60agatttatgc atttaacaca attcacagat caagtcaaca tccctatgtt tgtaagaagt 120attcctggat taagatggtg cagtacattc tttaactatc agattttaat gttagaaaat 180ttagcaccac aaggccctgc cgttttaaat cctcctttaa acggattgcc gcctcctcat 240ttgctgattg gctacgctta tctattcaat gtgaacaata actaccgttt tgaaagcaga 300acatatacaa aattaaacta tgaagctgat caatctacta caagaagacc tagaaatttt 360tggtctattc taagtgattg ttcatataca attaacacaa gtaatgtgcg caccattcca 420gaaaactacg aagataactt aaaccagttt caagaagaaa ttcttattaa tagaattaga 480gctgacattg aatctagaag taacatgcaa ggaacaggtg ttacattaca accagaagct 540tacgaaaata taaatatcca aaatgaaata aataaaatgt acgtaacaaa tttaagagat 600tttattaact ccaaaagttt tgctttcaat caaaggtatc aatatgaaac tgaaaaagat 660attaacacat taaagtgcat taactacact ttagaactac ttgcaaaatt tatttataat 720tggcaattta aagatgaaaa aatatacgta ccgttgaagg ataattggct tcaaatcctc 780aaaacagaat acgacaaatg gcagcctgaa atggatataa actacgctgt atcatatatc 840acagcattaa attcaatgat atttcctttc aaagagtgga aaacaaatct aaaaggtggt 900gcaagattaa gaagtggcac tagaacagac ttaccatttt taagacagag agaaaatcaa 960agagccataa ctgaacaaat gagaagaaat agaggacaga tagtttcgag gtttatagac 1020agcttacctt taatcagaag aataagaaga cccccaccaa gtcctgtgga agaggaagat 1080gcaggagaag gtccaagtgc cggacctgaa gaagaagaaa tggaagaaga attaggcaat 1140gaaatccttc gaatttttca aaatatcctt aatgaactaa gagcagaatt aacagaacct 1200gcaagagaac atgaaatttt ttctttcggt cagttatttt acaatctttt acagagagca 1260aatgaacaag gaagggtaac tagagagttt ataagaagat tcatatttta ctttttcata 1320gctgaacaca tcagtagtac actattttat tatcatgctc tattaaactt aaacgttatt 1380ttcagaagat atgtaaatat gcaatatgtc caagtcataa tgactggacg agatcatgaa 1440ggaaatgtta accttcacag agtttggact aacacaaaca tttctccatt tttaagaata 1500tttagaacaa tcatcaatga ccttctaata atatgtgaca gaaggccaga tagtattgaa 1560actcaagcag aacaagaaga cctacttaca tccctatcac acagaccaga atctggagat 1620ccaaacgacc ttctaaacca agctagactt aatgaagatt taatcaacac agtaactctt 1680tcctttaaaa tcaagcctat tggtcttgta acaattgcta caaacagaca aataattaac 1740aatgcttcgg ctgtaagaac acaagaaatg agaagattaa gacaaccaag a 1791 29 597 PRT hemorrhagic enteritis virus 29Met Ser Phe Phe Gln Ile Asn Asn Tyr Ala Arg Leu Thr Asn Gln Glu1 5 10 15Glu Asp Thr Ile Arg Phe Met His Leu Thr Gln Phe Thr Asp Gln Val 20 25 30Asn Ile Pro Met Phe Val Arg Ser Ile Pro Gly Leu Arg Trp Cys Ser 35 40 45Thr Phe Phe Asn Tyr Gln Ile Leu Met Leu Glu Asn Leu Ala Pro Gln 50 55 60Gly Pro Ala Val Leu Asn Pro Pro Leu Asn Gly Leu Pro Pro Pro His65 70 75 80Leu Leu Ile Gly Tyr Ala Tyr Leu Phe Asn Val Asn Asn Asn Tyr Arg 85 90 95Phe Glu Ser Arg Thr Tyr Thr Lys Leu Asn Tyr Glu Ala Asp Gln Ser 100 105 110Thr Thr Arg Arg Pro Arg Asn Phe Trp Ser Ile Leu Ser Asp Cys Ser 115 120 125Tyr Thr Ile Asn Thr Ser Asn Val Arg Thr Ile Pro Glu Asn Tyr Glu 130 135 140Asp Asn Leu Asn Gln Phe Gln Glu Glu Ile Leu Ile Asn Arg Ile Arg145 150 155 160Ala Asp Ile Glu Ser Arg Ser Asn Met Gln Gly Thr Gly Val Thr Leu 165 170 175Gln Pro Glu Ala Tyr Glu Asn Ile Asn Ile Gln Asn Glu Ile Asn Lys 180 185 190Met Tyr Val Thr Asn Leu Arg Asp Phe Ile Asn Ser Lys Ser Phe Ala 195 200 205Phe Asn Gln Arg Tyr Gln Tyr Glu Thr Glu Lys Asp Ile Asn Thr Leu 210 215 220Lys Cys Ile Asn Tyr Thr Leu Glu Leu Leu Ala Lys Phe Ile Tyr Asn225 230 235 240Trp Gln Phe Lys Asp Glu Lys Ile Tyr Val Pro Leu Lys Asp Asn Trp 245 250 255Leu Gln Ile Leu Lys Thr Glu Tyr Asp Lys Trp Gln Pro Glu Met Asp 260 265 270Ile Asn Tyr Ala Val Ser Tyr Ile Thr Ala Leu Asn Ser Met Ile Phe 275 280 285Pro Phe Lys Glu Trp Lys Thr Asn Leu Lys Gly Gly Ala Arg Leu Arg 290 295 300Ser Gly Thr Arg Thr Asp Leu Pro Phe Leu Arg Gln Arg Glu Asn Gln305 310 315 320Arg Ala Ile Thr Glu Gln Met Arg Arg Asn Arg Gly Gln Ile Val Ser 325 330 335Arg Phe Ile Asp Ser Leu Pro Leu Ile Arg Arg Ile Arg Arg Pro Pro 340 345 350Pro Ser Pro Val Glu Glu Glu Asp Ala Gly Glu Gly Pro Ser Ala Gly 355 360 365Pro Glu Glu Glu Glu Met Glu Glu Glu Leu Gly Asn Glu Ile Leu Arg 370 375 380Ile Phe Gln Asn Ile Leu Asn Glu Leu Arg Ala Glu Leu Thr Glu Pro385 390 395 400Ala Arg Glu His Glu Ile Phe Ser Phe Gly Gln Leu Phe Tyr Asn Leu 405 410 415Leu Gln Arg Ala Asn Glu Gln Gly Arg Val Thr Arg Glu Phe Ile Arg 420 425 430Arg Phe Ile Phe Tyr Phe Phe Ile Ala Glu His Ile Ser Ser Thr Leu 435 440 445Phe Tyr Tyr His Ala Leu Leu Asn Leu Asn Val Ile Phe Arg Arg Tyr 450 455 460Val Asn Met Gln Tyr Val Gln Val Ile Met Thr Gly Arg Asp His Glu465 470 475 480Gly Asn Val Asn Leu His Arg Val Trp Thr Asn Thr Asn Ile Ser Pro 485 490 495Phe Leu Arg Ile Phe Arg Thr Ile Ile Asn Asp Leu Leu Ile Ile Cys 500 505 510Asp Arg Arg Pro Asp Ser Ile Glu Thr Gln Ala Glu Gln Glu Asp Leu 515 520 525Leu Thr Ser Leu Ser His Arg Pro Glu Ser Gly Asp Pro Asn Asp Leu 530 535 540Leu Asn Gln Ala Arg Leu Asn Glu Asp Leu Ile Asn Thr Val Thr Leu545 550 555 560Ser Phe Lys Ile Lys Pro Ile Gly Leu Val Thr Ile Ala Thr Asn Arg 565 570 575Gln Ile Ile Asn Asn Ala Ser Ala Val Arg Thr Gln Glu Met Arg Arg 580 585 590Leu Arg Gln Pro Arg 595 30 1038 DNA hemorrhagic enteritis virus 30atgcaatacg ctacaggatt acaagcattg tttgcatgcc aagctgaagc gaatatttta 60cctacttctg attactgggc caaactggtt gagacatatg ttaagaagaa taaaccagat 120cttaatctta cgatttcctc cgctaaatct ttttatcact ttgctggccg tatcgtaagc 180agttttgtgt ataatgatag cggcttaatt tgcaattata attgccttgg agccaatatc 240tgggttcata attgggatga agataacatt agatgttttc atggcactgt tatgctttca 300aaacccataa cttacagcct gtctcctcaa tcagaggaag gaatgagagc tctgacagct 360ggtgagggca gattagagaa agaaaaaaat caaaaagacg tgataaagct aacaaattat 420agcaatatag tctgtccatt agatagtaat gttcagtggc caactattca ttcaccagac 480tcttgtggaa tgaattttgg caacaaagaa aaagcaaaag ctgcctttct tcataatatt 540gattggacaa gtgctatgtt tcctaaagca aagaaatgtg aaatttctga aaaaatgatt 600atagttacaa agtgcttctg taactatggt catgaaaaca ttcagcttgg cagacaaatc 660tgcaaaatga ctgcttttga aatacctgga gcgaatgaca tagaccctga gagttgtcat 720gatgatatgc ttcttgcaac tgccaagtat aaacacacct ttgtgtttca atgttgcaat 780cccattagac ttaaaagaaa tgctaaggat aaagataatc agactcacaa acattgtgat 840tttaaattgt caatgattga tgtgagacag gctatgaaaa ttagcaaaga tatatatact 900aagctgaagg aaaccataga tgatggctcc cctactaaaa taatgttgcc tgcttttgta 960ttcaatccta aaaagcattc ttttaagcaa gctattgtgg ctcaacacga ggtggaaagt 1020gatgatgacg ctttttgt 1038 31 346 PRT hemorrhagic enteritis virus 31Met Gln Tyr Ala Thr Gly Leu Gln Ala Leu Phe Ala Cys Gln Ala Glu1 5 10 15Ala Asn Ile Leu Pro Thr Ser Asp Tyr Trp Ala Lys Leu Val Glu Thr 20 25 30Tyr Val Lys Lys Asn Lys Pro Asp Leu Asn Leu Thr Ile Ser Ser Ala 35 40 45Lys Ser Phe Tyr His Phe Ala Gly Arg Ile Val Ser Ser Phe Val Tyr 50 55 60Asn Asp Ser Gly Leu Ile Cys Asn Tyr Asn Cys Leu Gly Ala Asn Ile65 70 75 80Trp Val His Asn Trp Asp Glu Asp Asn Ile Arg Cys Phe His Gly Thr 85 90 95Val Met Leu Ser Lys Pro Ile Thr Tyr Ser Leu Ser Pro Gln Ser Glu 100 105 110Glu Gly Met Arg Ala Leu Thr Ala Gly Glu Gly Arg Leu Glu Lys Glu 115 120 125Lys Asn Gln Lys Asp Val Ile Lys Leu Thr Asn Tyr Ser Asn Ile Val 130 135 140Cys Pro Leu Asp Ser Asn Val Gln Trp Pro Thr Ile His Ser Pro Asp145 150 155 160Ser Cys Gly Met Asn Phe Gly Asn Lys Glu Lys Ala Lys Ala Ala Phe 165 170 175Leu His Asn Ile Asp Trp Thr Ser Ala Met Phe Pro Lys Ala Lys Lys 180 185 190Cys Glu Ile Ser Glu Lys Met Ile Ile Val Thr Lys Cys Phe Cys Asn 195 200 205Tyr Gly His Glu Asn Ile Gln Leu Gly Arg Gln Ile Cys Lys Met Thr 210 215 220Ala Phe Glu Ile Pro Gly Ala Asn Asp Ile Asp Pro Glu Ser Cys His225 230 235 240Asp Asp Met Leu Leu Ala Thr Ala Lys Tyr Lys His Thr Phe Val Phe 245 250 255Gln Cys Cys Asn Pro Ile Arg Leu Lys Arg Asn Ala Lys Asp Lys Asp 260 265 270Asn Gln Thr His Lys His Cys Asp Phe Lys Leu Ser Met Ile Asp Val 275 280 285Arg Gln Ala Met Lys Ile Ser Lys Asp Ile Tyr Thr Lys Leu Lys Glu 290 295 300Thr Ile Asp Asp Gly Ser Pro Thr Lys Ile Met Leu Pro Ala Phe Val305 310 315 320Phe Asn Pro Lys Lys His Ser Phe Lys Gln Ala Ile Val Ala Gln His 325 330 335Glu Val Glu Ser Asp Asp Asp Ala Phe Cys 340 345 32 7 PRT hemorrhagic enteritis virus 32Val Phe Gln Cys Cys Asn Pro1 5 33 12 PRT adeno-associated virus 2 33Arg Leu Pro Val Arg Arg Arg Arg Arg Arg Val Pro1 5 10 34 12 PRT EDS 34Thr Leu Pro Ala Arg Thr Arg Arg Thr Arg Arg Pro1 5 10 35 12 PRT hemorrhagic enteritis virus 35Ser Leu Pro Leu Ile Arg Arg Ile Arg Arg Pro Pro1 5 10 36 5 PRT hemorrhagic enteritis virus 36Leu Arg Gly Gly Lys1 5 37 11 PRT Unknown Description of Unknown Organism Conserved sequence 37Gly Xaa Xaa Xaa Xaa Xaa Arg Arg Xaa Cys Xaa1 5 10 38 7 PRT adeno-associated virus 2 MISC_FEATURE (4)..(4) X=Tyr or Arg or Phe 38Met Leu Ile Xaa Gly Xaa Gly1 5 39 7 PRT adeno-associated virus 2 MISC_FEATURE (4)..(4) X=Tyr or Arg or Phe 39Met Leu Ile Xaa Gly Gly Xaa1 5 40 6 PRT hemorrhagic enteritis virus 40Gly Gly Gly Asn Gly Gly1 5 41 4 PRT hemorrhagic enteritis virus 41Lys Lys Arg Lys1 42 4 PRT hemorrhagic enteritis virus 42Lys Lys Asn Lys1

Claims

1. An isolated DNA sequence which comprises:a) a nucleotide sequence as shown in SEQ ID NO:1; or b) a nucleotide sequence which corresponds to the sequence shown in SEQ ID NO:1 within the scope of the degeneracy of the genetic code.

2. The isolated DNA sequence as claimed in claim 1 which is genomic DNA or cDNA.

3. A non-virulent HEV encoded by the DNA sequence of claim 1.

4. A host cell transformed with a nucleotide sequence which comprises:a) a nucleotide sequence as shown in SEQ ID NO:1; or b) a nucleotide sequence which corresponds to the sequence shown in SEQ ID NO:1 within the scope of the degeneracy of the genetic code.

5. A host cell as claimed in claim 4, said host cell being a eukaryotic host cell, wherein said eukaryotic host cell is an insect cell, a plant cell, a mammalian cell, a bird cell, or a yeast cell, or a prokaryotic host cell.

6. A process for the production of transformed or transfected host cells as claimed in claim 4, which process comprises:a) transforming a host cell with a DNA sequence which comprises: i) a nucleotide sequence as shown in SEQ ID NO:1; or ii) a nucleotide sequence which corresponds to the sequence shown in SEQ ID NO:1 within the scope of the degeneracy of the genetic code; b) isolating the transformed or transfected cells obtained by step (a); and c) culturing the host cell isolated in step (b) under conditions in which propagation of said cells takes place.

7. A method of eliciting an immune response in an animal against Hemorrhagic enteritis virus (HEV) by administering to the animal the nucleotide sequence as claimed in claim 1.

8. The host cell of claim 5, wherein the prokaryotic host cell is a bacterial cell.

9. The pharmaceutical composition comprising as active ingredient a therapeutically effective amount of the cells of claim 4, and a pharmaceutically acceptable carrier.